Method, apparatus and computer program product for determining a set of non-hybridizing oligonucleotides

ABSTRACT

The invention provides a computerized method, associated apparatus, and computer program product for determining a set of non-hybridizing oligonucleotides. The invention represents a first oligonucleotide in the computer&#39;s memory, generates a set of oligonucleotides, including the first oligonucleotide, that meet a specified condition that determines whether the generated oligonucleotides will not hybridize with the first oligonucleotide. The invention also examines each of the generated oligonucleotides in the set to remove oligonucleotides from the set that hybridize with other nucleotides in the set. Thus, the invention develops a minimally cross-hybridizing set of oligonucleotides that can be used for tracking, identifying, and/or sorting classes or subpopulations of molecules by the user of oligonucleotide tags.

This is a divisional of Ser. No. 08/659,453 filed Jun. 6, 1996, now U.S.Pat. No. 5,846,719, which is a continuation-in-part of Ser. No.08/358,810 filed Dec. 19, 1994, now U.S. Pat. No. 5,604,097, which is acontinuation-in-part of Ser. No. 08/322,348 filed Oct. 13, 1994, nowabandoned, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of computerized methods fordetermining non-hybridizing oligonucleotides. Specifically, thisinvention includes a method, apparatus, and computer program product fordetermining a non-hybridizing set of oligonucleotides having specifiedcharacteristics.

BACKGROUND

Specific hybridization of oligonucleotides and their analogs is afundamental process that is employed in a wide variety of research,medical, and industrial applications, including the identification ofdisease-related polynucleotides in diagnostic assays, screening forclones of novel target polynucleotides, identification of specificpolynucleotides in blots of mixtures of polynucleotides, amplificationof specific target polynucleotides, therapeutic blocking ofinappropriately expressed genes, DNA sequencing, and the like, e.g.Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition(Cold Spring Harbor Laboratory, New York, 1989); Keller and Manak, DNAProbes, 2nd Edition (Stockton Press, New York, 1993); Milligan et al, J.Med. Chem., 36: 1923-1937 (1993); Drmanac et al, Science, 260: 1649-1652(1993); Bains, J. DNA Sequencing and Mapping, 4: 143-150 (1993).

Specific hybridization has also been proposed as a method of tracking,retrieving, and identifying compounds labeled with oligonucleotide tags.For example, in multiplex DNA sequencing oligonucleotide tags are usedto identify electrophoretically separated bands on a gel that consist ofDNA fragments generated in the same sequencing reaction. In this way,DNA fragments from many sequencing reactions are separated on the samelane of a gel which is then blotted with separate solid phase materialson which the fragment bands from the separate sequencing reactions arevisualized with oligonucleotide probes that specifically hybridize tocomplementary tags, Church et al, Science, 240: 185-188 (1988). Similaruses of oligonucleotide tags have also been proposed for identifyingexplosives, potential pollutants, such as crude oil, and currency forprevention and detection of counterfeiting, e.g. reviewed by Dollinger,pages 265-274 in Mullis et al, editors, The Polymerase Chain Reaction(Birkhauser, Boston, 1994). More recently, systems employingoligonucleotide tags have also been proposed as a means of manipulatingand identifying individual molecules in complex combinatorial chemicallibraries, for example, as an aid to screening such libraries for drugcandidates, Brenner and Lerner, Proc. Natl. Acad. Sci., 89: 5381-5383(1992); Alper, Science, 264: 1399-1401 (1994); and Needels et al, Proc.Natl. Acad. Sci., 90: 10700-10704 (1993).

The successful implementation of such tagging schemes depends in largepart on the success in achieving specific hybridization between a tagand its complementary probe. That is, for an oligonucleotide tag tosuccessfully identify a substance, the number of false positive andfalse negative signals must be minimized. Unfortunately, such spurioussignals are not uncommon because base pairing and base stacking freeenergies vary widely among nucleotides in a duplex or triplex structure.For example, a duplex consisting of a repeated sequence ofdeoxyadenosine (A) and thymidine (T) bound to its complement may haveless stability than an equal-length duplex consisting of a repeatedsequence of deoxyguanosine (G) and deoxycytidine (C) bound to apartially complementary target containing a mismatch. Thus, if a desiredcompound from a large combinatorial chemical library were tagged withthe former oligonucleotide, a significant possibility would exist that,under hybridization conditions designed to detect perfectly matchedAT-rich duplexes, undesired compounds labeled with the GC-richoligonucleotide--even in a mismatched duplex--would be detected alongwith the perfectly matched duplexes consisting of the AT-rich tag. Inthe molecular tagging system proposed by Brenner et al (cited above),the related problem of mis-hybridizations of closely related tags wasaddressed by employing a so-called "comma-less" code, which ensures thata probe out of register (or frame shifted) with respect to itscomplementary tag would result in a duplex with one or more mismatchesfor each of its five or more three-base words, or "codons."

Even though reagents, such as tetramethylammonium chloride, areavailable to negate base-specific stability differences ofoligonucleotide duplexes, the effect of such reagents is often limitedand their presence can be incompatible with, or render more difficult,further manipulations of the selected compounds, e.g. amplification bypolymerase chain reaction (PCR), or the like.

Such problems have made the simultaneous use of multiple hybridizationprobes in the analysis of multiple or complex genetic loci, e.g. viamultiplex PCR, reverse dot blotting, or the like, very difficult. As aresult, direct sequencing of certain loci, e.g. HLA genes, has beenpromoted as a reliable alternative to indirect methods employingspecific hybridization for the identification of genotypes, e.g.Gyllensten et al, Proc. Natl. Acad. Sci., 85: 7652-7656 (1988).

The ability to sort cloned and identically tagged DNA fragments ontodistinct solid phase supports would facilitate such sequencing,particularly when coupled with a non gel-based sequencing methodologysimultaneously applicable to many samples in parallel.

In view of the above, it would be useful if there were available anoligonucleotide-based tagging system which provided a large repertoireof tags, but which also minimized the occurrence of false positive andfalse negative signals without the need to employ special reagents foraltering natural base pairing and base stacking free energy differences.Such a tagging system would find applications in many areas, includingconstruction and use of combinatorial chemical libraries, large-scalemapping and sequencing of DNA, genetic identification, medicaldiagnostics, and the like.

SUMMARY OF THE INVENTION

An object of my invention is to provide a molecular tagging system fortracking, retrieving, and identifying compounds.

Another object of my invention is to provide a method for sortingidentical molecules, or subclasses of molecules, especiallypolynucleotides, onto surfaces of solid phase materials by the specifichybridization of oligonucleotide tags and their complements.

A further object of my invention is to provide a method for analyzinggene expression patterns in diseased and normal tissues.

A still further object of my invention is to provide a system fortagging and sorting many thousands of fragments, especially randomlyoverlapping fragments, of a target polynucleotide for simultaneousanalysis and/or sequencing.

Another object of my invention is to provide a rapid and reliable methodfor sequencing target polynucleotides having a length in the range of afew hundred basepairs to several tens of thousands of basepairs.

A further object of my invention is to provide a method for reducing thenumber of separate template preparation steps required in large scalesequencing projects employing conventional Sanger-based sequencingtechniques.

My invention achieves these and other objects by providing a method andmaterials for tracking, identifying, and/or sorting classes orsubpopulations of molecules by the use of oligonucleotide tags. Animportant feature of the invention is that the oligonucleotide tags aremembers of a minimally cross-hybridizing set of oligonucleotides. Thesequences of oligonucleotides of such a set differ from the sequences ofevery other member of the same set by at least two nucleotides. Thus,each member of such a set cannot form a duplex (or triplex) with thecomplement of any other member with less than two mismatches.Complements of oligonucleotide tags of the invention, referred to hereinas "tag complements," may comprise natural nucleotides or non-naturalnucleotide analogs. Preferably, tag complements are attached to solidphase supports. Such oligonucleotide tags when used with theircorresponding tag complements provide a means of enhancing specificityof hybridization for sorting, tracking, or labeling molecules,especially polynucleotides.

Minimally cross-hybridizing sets of oligonucleotide tags and tagcomplements may be synthesized either combinatorially or individuallydepending on the size of the set desired and the degree to whichcross-hybridization is sought to be minimized (or stated another way,the degree to which specificity is sought to be enhanced). For example,a minimally cross-hybridizing set may consist of a set of individuallysynthesized 10-mer sequences that differ from each other by at least 4nucleotides, such set having a maximum size of 332 (when composed of 3kinds of nucleotides and counted using a computer program such asdisclosed in Appendix Ic). Alternatively, a minimally cross-hybridizingset of oligonucleotide tags may also be assembled combinatorially fromsubunits which themselves are selected from a minimallycross-hybridizing set. For example, a set of minimally cross-hybridizing12-mers differing from one another by at least three nucleotides may besynthesized by assembling 3 subunits selected from a set of minimallycross-hybridizing 4-mers that each differ from one another by threenucleotides. Such an embodiment gives a maximally sized set of 9³, or729, 12-mers. The number 9 is number of oligonucleotides listed by thecomputer program of Appendix Ia, which assumes, as with the 10-mers,that only 3 of the 4 different types of nucleotides are used. The set isdescribed as "maximal" because the computer programs of Appendices Ia-cprovide the largest set for a given input (e.g. length, composition,difference in number of nucleotides between members). Additionalminimally cross-hybridizing sets may be formed from subsets of suchcalculated sets.

Oligonucleotide tags may be single stranded and be designed for specifichybridization to single stranded tag complements by duplex formation orfor specific hybridization to double stranded tag complements by triplexformation. Oligonucleotide tags may also be double stranded and bedesigned for specific hybridization to single stranded tag complementsby triplex formation.

When synthesized combinatorially, an oligonucleotide tag of theinvention preferably consists of a plurality of subunits, each subunitconsisting of an oligonucleotide of 3 to 9 nucleotides in length whereineach subunit is selected from the same minimally cross-hybridizing set.In such embodiments, the number of oligonucleotide tags availabledepends on the number of subunits per tag and on the length of thesubunits. The number is generally much less than the number of allpossible sequences the length of the tag, which for a tag n nucleotideslong would be 4^(n).

In one aspect of my invention, complements of oligonucleotide tagsattached to a solid phase support are used to sort polynucleotides froma mixture of polynucleotides each containing a tag. In this embodiment,complements of the oligonucleotide tags are synthesized on the surfaceof a solid phase support, such as a microscopic bead or a specificlocation on an array of synthesis locations on a single support, suchthat populations of identical sequences are produced in specificregions. That is, the surface of each support, in the case of a bead, orof each region, in the case of an array, is derivatized by only one typeof complement which has a particular sequence. The population of suchbeads or regions contains a repertoire of complements with distinctsequences. As used herein in reference to oligonucleotide tags and tagcomplements, the term "repertoire" means the set of minimallycross-hybridizing set of oligonucleotides that make up the tags in aparticular embodiment or the corresponding set of tag complements.

The polynucleotides to be sorted each have an oligonucleotide tagattached, such that different polynucleotides have different tags. Asexplained more fully below, this condition is achieved by employing arepertoire of tags substantially greater than the population ofpolynucleotides and by taking a sufficiently small sample of taggedpolynucleotides from the full ensemble of tagged polynucleotides. Aftersuch sampling, when the populations of supports and polynucleotides aremixed under conditions which permit specific hybridization of theoligonucleotide tags with their respective complements, identicalpolynucleotides sort onto particular beads or regions. The sortedpopulations of polynucleotides can then be manipulated on the solidphase support by micro-biochemical techniques.

Generally, the method of my invention comprises the following steps: (a)attaching an oligonucleotide tag from a repertoire of tags to eachmolecule in a population of molecules (i) such that substantially alldifferent molecules or different subpopulations of molecules in thepopulation have different oligonucleotide tags attached and (ii) suchthat each oligonucleotide tag from the repertoire is selected from thesame minimally cross-hybridizing set; and (b) sorting the molecules ofthe population onto one or more solid phase supports by specificallyhybridizing the oligonucleotide tags with their respective complementsattached to such supports.

An important aspect of my invention is the use of the oligonucleotidetags to sort polynucleotides for parallel sequence determination.Preferably, such sequencing is carried out by the following steps: (a)generating from the target polynucleotide a plurality of fragments thatcover the target polynucleotide; (b) attaching an oligonucleotide tagfrom a repertoire of tags to each fragment of the plurality (i) suchsubstantially all different fragments have different oligonucleotidetags attached and (ii) such that each oligonucleotide tag from therepertoire is selected from the same minimally cross-hybridizing set;(c) sorting the fragments onto one or more solid phase supports byspecifically hybridizing the oligonucleotide tags with their respectivecomplements attached to the solid phase supports; (d) determining thenucleotide sequence of a portion of each of the fragments of theplurality, preferably by a single-base sequencing methodology asdescribed below; and (e) determining the nucleotide sequence of thetarget polynucleotide by collating the sequences of the fragments.

Another important aspect of my invention is the determination of aprofile, or a frequency distribution, of genes being expressed in agiven tissue or cell type, wherein each such gene is identified by aportion of its sequence. Preferably, such frequency distribution isdetermined by the following steps: (a) forming a cDNA library from apopulation of mRNA molecules, each cDNA molecule in the cDNA libraryhaving an oligonucleotide tag attached, (i) such that substantially alldifferent cDNA molecules have different oligonucleotide tags attachedand (ii) such that each oligonucleotide tag from the repertoire isselected from the same minimally cross-hybridizing set; (b) sorting thecDNA molecules by specifically hybridizing the oligonucleotide tags withtheir respective complements attached to one or more solid phasesupports; (c) determining the nucleotide sequence of a portion of eachof the sorted cDNA molecules; and (d) forming a frequency distributionof mRNA molecules from the nucleotide sequences of the portions ofsorted cDNA molecules.

My invention overcomes a key deficiency of current methods of tagging orlabeling molecules with oligonucleotides: By coding the sequences of thetags in accordance with the invention, the stability of any mismatchedduplex or triplex between a tag and a complement to another tag is farlower than that of any perfectly matched duplex between the tag and itsown complement. Thus, the problem of incorrect sorting because ofmismatch duplexes of GC-rich tags being more stable than perfectlymatched AT-rich tags is eliminated.

When used in combination with solid phase supports, such as microscopicbeads, my invention provides a readily automated system for manipulatingand sorting polynucleotides, particularly useful in large-scale paralleloperations, such as large-scale DNA sequencing, wherein many targetpolynucleotides or many segments of a single target polynucleotide aresequenced and/or analyzed simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating a general algorithm for generatingminimally cross-hybridizing sets.

FIG. 2 diagrammatically illustrates an apparatus for carrying outparallel operations, such as polynucleotide sequencing, in accordancewith the invention.

FIG. 3 illustrates an embodiment for genotyping by sorting ligatedprobes onto a solid phase support.

DEFINITIONS

"Complement" or "tag complement" as used herein in reference tooligonucleotide tags refers to an oligonucleotide to which aoligonucleotide tag specifically hybridizes to form a perfectly matchedduplex or triplex. In embodiments where specific hybridization resultsin a triplex, the oligonucleotide tag may be selected to be eitherdouble stranded or single stranded. Thus, where triplexes are formed,the term "complement" is meant to encompass either a double strandedcomplement of a single stranded oligonucleotide tag or a single strandedcomplement of a double stranded oligonucleotide tag.

The term "oligonucleotide" as used herein includes linear oligomers ofnatural or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptidenucleic acids (PNAs), and the like, capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like.Usually monomers are linked by phosphodiester bonds or analogs thereofto form oligonucleotides ranging in size from a few monomeric units,e.g. 3-4, to several tens of monomeric units. Whenever anoligonucleotide is represented by a sequence of letters, such as"ATGCCTG," it will be understood that the nucleotides are in 5'→3' orderfrom left to right and that "A" denotes deoxyadenosine, "C" denotesdeoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine,unless otherwise noted. Analogs of phosphodiester linkages includephosphorothioate, phosphorodithioate, phosphoranilidate,phosphoramidate, and the like. Usually oligonucleotides of the inventioncomprise the four natural nucleotides; however, they may also comprisenon-natural nucleotide analogs. It is clear to those skilled in the artwhen oligonucleotides having natural or non-natural nucleotides may beemployed, e.g. where processing by enzymes is called for, usuallyoligonucleotides consisting of natural nucleotides are required.

"Perfectly matched" in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one other such that every nucleotide in each strandundergoes Watson-Crick basepairing with a nucleotide in the otherstrand. The term also comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, and thelike, that may be employed. In reference to a triplex, the term meansthat the triplex consists of a perfectly matched duplex and a thirdstrand in which every nucleotide undergoes Hoogsteen or reverseHoogsteen association with a basepair of the perfectly matched duplex.Conversely, a "mismatch" in a duplex between a tag and anoligonucleotide means that a pair or triplet of nucleotides in theduplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/orreverse Hoogsteen bonding.

As used herein, "nucleoside" includes the natural nucleosides, including2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90: 543-584 (1990), or the like, with the only provisothat they are capable of specific hybridization. Such analogs includesynthetic nucleosides designed to enhance binding properties, reducecomplexity, increase specificity, and the like.

As used herein "sequence determination" or "determining a nucleotidesequence" in reference to polynucleotides includes determination ofpartial as well as full sequence information of the polynucleotide. Thatis, the term includes sequence comparisons, fingerprinting, and likelevels of information about a target polynucleotide, as well as theexpress identification and ordering of nucleosides, usually eachnucleoside, in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. For example, in some embodiments sequence determinationmay be effected by identifying the ordering and locations of a singletype of nucleotide, e.g. cytosines, within the target polynucleotide"CATCGC . . ." so that its sequence is represented as a binary code,e.g. "100101 . . . " for "C-(not C)-(not C)-C-(not C)-C . . . " and thelike.

As used herein, the term "complexity" in reference to a population ofpolynucleotides means the number of different species of moleculepresent in the population.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of labeling and sorting molecules,particularly polynucleotides, by the use of oligonucleotide tags. Theoligonucleotide tags of the invention belong to minimallycross-hybridizing sets of oligonucleotides. Thus, the sequences of anytwo oligonucleotide tags of a repertoire will never be "closer" thandiffering by two nucleotides. In particular embodiments, sequences ofany two oligonucleotide tags of a repertoire can be even "further"apart, e.g. by designing a minimally cross-hybridizing set such thatoligonucleotides cannot form a duplex or triplex with the complement ofanother member of the same set with less than three mismatchednucleotides, and so on. In such embodiments, greater specificity isachieved, but the total repertoire of tags is smaller. Thus, for tags ofa given length, a trade off must be made between the degree ofspecificity desired and the size of repertoire desired. The invention isparticularly useful in labeling and sorting polynucleotides for paralleloperations, such as sequencing, fingerprinting or other types ofanalysis.

Oligonucleotide Tags and Tag Complements

The nucleotide sequences of oligonucleotides of a minimallycross-hybridizing set are conveniently enumerated by simple computerprograms following the general algorithm illustrated in FIG. 1, and asexemplified by programs whose source codes are listed in Appendices Iaand Ib. Program minhx of Appendix Ia computes all minimallycross-hybridizing sets having 4-mer subunits composed of three kinds ofnucleotides. Program tagN of Appendix Ib enumerates longeroligonucleotides of a minimally cross-hybridizing set. Similaralgorithms and computer programs are readily written for listingoligonucleotides of minimally cross-hybridizing sets for any embodimentof the invention. Table I below provides guidance as to the size of setsof minimally cross-hybridizing oligonucleotides for the indicatedlengths and number of nucleotide differences. The above computerprograms were used to generate the numbers.

                                      TABLE I                                     __________________________________________________________________________            Nucleotide                                                                    Difference                                                                             Maximal Size                                                         between  of Minimally                                                                         Size of                                               Oligonucleotide                                                                       Oligonucleotides                                                                       Cross- Repertoire                                                                          Size of                                         Word    of Minimally                                                                           Hybridizing                                                                          with Four                                                                           Repertoire with                                 Length  Hybridizing Set                                                                        Set    Words Five Words                                      __________________________________________________________________________    4       3        9      6561  5.90 × 10.sup.4                           6       3        27     5.3 × 10.sup.5                                                                1.43 × 10.sup.7                           7       4        27     5.3 × 10.sup.5                                                                1.43 × 10.sup.7                           7       5        8      4096  3.28 × 10.sup.4                           8       3        190    1.30 × 10.sup.9                                                               2.48 × 10.sup.11                          8       4        62     1.48 × 10.sup.7                                                               9.16 × 10.sup.8                           8       5        18     1.05 × 10.sup.5                                                               1.89 × 10.sup.6                           9       5        39     2.31 × 10.sup.6                                                               9.02 × 10.sup.7                           10      5        332    1.21 × 10.sup.10                                10      6        28     6.15 × 10.sup.5                                                               1.72 × 10.sup.7                           11      5        187                                                          18      6        ≈ 25000                                              18      12       24                                                           __________________________________________________________________________

For some embodiments of the invention, where extremely large repertoiresof tags are not required, oligonucleotide tags of a minimallycross-hybridizing set may be separately synthesized. Sets containingseveral hundred to several thousands, or even several tens of thousands,of oligonucleotides may be synthesized directly by a variety of parallelsynthesis approaches, e.g. as disclosed in Frank et al, U.S. Pat. No.4,689,405; Frank et al, Nucleic Acids Research, 11: 4365-4377 (1983);Matson et al, Anal. Biochem., 224: 110-116 (1995); Fodor et al,International application PCT/US93/04145; Pease et al, Proc. Natl. Acad.Sci., 91: 5022-5026 (1994); Southern et al, J. Biotechnology, 35:217-227 (1994), Brennan, International application PCT/US94/05896;Lashkari et al, Proc. Natl. Acad. Sci., 92: 7912-7915 (1995); or thelike.

Preferably, oligonucleotide tags of the invention are synthesizedcombinatorially out of subunits between three and six nucleotides inlength and selected from the same minimally cross-hybridizing set. Foroligonucleotides in this range, the members of such sets may beenumerated by computer programs based on the algorithm of FIG. 1.

The algorithm of FIG. 1 is implemented by first defining thecharacteristics of the subunits of the minimally cross-hybridizing set,i.e. length, number of base differences between members, andcomposition, e.g. do they consist of two, three, or four kinds of bases.A table M_(n), n=1, is generated (100) that consists of all possiblesequences of a given length and composition. An initial subunit S₁ isselected and compared (120) with successive subunits S_(i) for i=n+1 tothe end of the table. Whenever a successive subunit has the requirednumber of mismatches to be a member of the minimally cross-hybridizingset, it is saved in a new table M_(n+1) (125), that also containssubunits previously selected in prior passes through step 120. Forexample, in the first set of comparisons, M₂ will contain S₁ ; in thesecond set of comparisons, M₃ will contain S₁ and S₂ ; in the third setof comparisons, M₄ will contain S₁, S₂, and S₃ ; and so on. Similarly,comparisons in table M_(j) will be between S_(j) and all successivesubunits in M_(j). Note that each successive table M_(n+1) is smallerthan its predecessors as subunits are eliminated in successive passesthrough step 130. After every subunit of table M_(n) has been compared(140) the old table is replaced by the new table M_(n+1), and the nextround of comparisons are begun. The process stops (160) when a tableM_(n) is reached that contains no successive subunits to compare to theselected subunit S_(i), i.e. M_(n) =M_(n+1).

Preferably, minimally cross-hybridizing sets comprise subunits that makeapproximately equivalent contributions to duplex stability as everyother subunit in the set. In this way, the stability of perfectlymatched duplexes between every subunit and its complement isapproximately equal. Guidance for selecting such sets is provided bypublished techniques for selecting optimal PCR primers and calculatingduplex stabilities, e.g. Rychlik et al, Nucleic Acids Research, 17:8543-8551 (1989) and 18: 6409-6412 (1990); Breslauer et al, Proc. Natl.Acad. Sci., 83: 3746-3750 (1986); Wetmur, Crit. Rev. Biochem. Mol.Biol., 26: 227-259 (1991); and the like. For shorter tags, e.g. about 30nucleotides or less, the algorithm described by Rychlik and Wetmur ispreferred, and for longer tags, e.g. about 30-35 nucleotides or greater,an algorithm disclosed by Suggs et al, pages 683-693 in Brown, editor,ICN-UCLA Symp. Dev. Biol., Vol. 23 (Academic Press, New York, 1981) maybe conveniently employed. Clearly, the are many approaches available toone skilled in the art for designing sets of minimally cross-hybridizingsubunits within the scope of the invention. For example, to minimize theaffects of different base-stacking energies of terminal nucleotides whensubunits are assembled, subunits may be provided that have the sameterminal nucleotides. In this way, when subunits are linked, the sum ofthe base-stacking energies of all the adjoining terminal nucleotideswill be the same, thereby reducing or eliminating variability in tagmelting temperatures.

A "word" of terminal nucleotides, shown in italic below, may also beadded to each end of a tag so that a perfect match is always formedbetween it and a similar terminal "word" on any other tag complement.Such an augmented tag would have the form:

    ______________________________________                                        W      W.sub.1  W.sub.2                                                                              . . .  W.sub.k-1                                                                           W.sub.k                                                                              W                                  W'     W.sub.1 '                                                                              W.sub.2 '                                                                            . . .  W.sub.k-1 '                                                                         W.sub.k '                                                                            W'                                 ______________________________________                                    

where the primed W's indicate complements. With ends of tags alwaysforming perfectly matched duplexes, all mismatched words will beinternal mismatches thereby reducing the stability of tag-complementduplexes that otherwise would have mismatched words at their ends. It iswell known that duplexes with internal mismatches are significantly lessstable than duplexes with the same mismatch at a terminus.

A preferred embodiment of minimally cross-hybridizing sets are thosewhose subunits are made up of three of the four natural nucleotides. Aswill be discussed more fully below, the absence of one type ofnucleotide in the oligonucleotide tags permits target polynucleotides tobe loaded onto solid phase supports by use of the 3'→3' exonucleaseactivity of a DNA polymerase. The following is an exemplary minimallycross-hybridizing set of subunits each comprising four nucleotidesselected from the group consisting of A, G, and T:

                  TABLE II                                                        ______________________________________                                           Word:     w1       w2       w3     w4                                      Sequence:      GATT   TGAT     TAGA   TTTG                                       Word:         W5     W6       W7     W8                                    Sequence:      GTAA   AGTA     ATGT   AAAG                                    ______________________________________                                    

In this set, each member would form a duplex having three mismatchedbases with the complement of every other member.

Further exemplary minimally cross-hybridizing sets are listed below inTable III. Clearly, additional sets can be generated by substitutingdifferent groups of nucleotides, or by using subsets of known minimallycross-hybridizing sets.

                  TABLE III                                                       ______________________________________                                        Exemplary Minimally Cross-Hybridizing Sets of 4-mer - Subunits                ______________________________________                                        Set 1   Set 2    Set 3    Set 4  Set 5  Set 6                                 ______________________________________                                        CATT     ACCC     AAAC     AAAG   AACA   AACG                                 CTAA     AGGG     ACCA     ACCA   ACAC   ACAA                                 TCAT     CACG     AGGG     AGGC   AGGG   AGGC                                 ACTA     CCGA     CACG     CACC   CAAG   CAAC                                 TACA     CGAC     CCGC     CCGG   CCGC   CCGG                                 TTTC     GAGC     CGAA     CGAA   CGCA   CGCA                                 ATCT     GCAG     GAGA     GAGA   GAGA   GAGA                                 AAAC     GGCA     GCAG     GCAC   GCCG   GCCC                                          AAAA     GGCC     GGCG   GGAC   GGAG                                 ______________________________________                                        Set 7   Set 8    Set 9    Set 10 Set 11 Set 12                                ______________________________________                                        AAGA     AAGC     AAGG     ACAG   ACCG   ACGA                                 ACAC     ACAA     ACAA     AACA   AAAA   AAAC                                 AGCG     AGCG     AGCC     AGGC   AGGC   AGCG                                 CAAG     CAAG     CAAC     CAAC   CACC   CACA                                 CCCA     CCCC     CCCG     CCGA   CCGA   CCAG                                 CGGC     CGGA     CGGA     CGCG   CGAG   CGGC                                 GACC     GACA     GACA     GAGG   GAGG   GAGG                                 GCGG     GCGG     GCGC     GCCC   GCAC   GCCC                                 GGAA     GGAC     GGAG     GGAA   GGCA   GGAA                                 ______________________________________                                    

The oligonucleotide tags of the invention and their complements areconveniently synthesized on an automated DNA synthesizer, e.g. anApplied Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNASynthesizer, using standard chemistries, such as phosphoramiditechemistry, e.g. disclosed in the following references: Beaucage andIyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. Pat. No.4,980,460; Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S.Pat. Nos. 4,415,732; 4,458,066; and 4,973,679; and the like. Alternativechemistries, e.g. resulting in non-natural backbone groups, such asphosphorothioate, phosphoramidate, and the like, may also be employedprovided that the resulting oligonucleotides are capable of specifichybridization. In some embodiments, tags may comprise naturallyoccurring nucleotides that permit processing or manipulation by enzymes,while the corresponding tag complements may comprise non-naturalnucleotide analogs, such as peptide nucleic acids, or like compounds,that promote the formation of more stable duplexes during sorting.

When microparticles are used as supports, repertoires of oligonucleotidetags and tag complements may be generated by subunit-wise synthesis via"split and mix" techniques, e.g. as disclosed in Shortle et al,International patent application PCT/US93/03418 or Lyttle et al,Biotechniques, 19: 274-280 (1995). Briefly, the basic unit of thesynthesis is a subunit of the oligonucleotide tag. Preferably,phosphoramidite chemistry is used and 3' phosphoramiditeoligonucleotides are prepared for each subunit in a minimallycross-hybridizing set, e.g. for the set first listed above, there wouldbe eight 4-mer 3'-phosphoramidites. Synthesis proceeds as disclosed byShortle et al or in direct analogy with the techniques employed togenerate diverse oligonucleotide libraries using nucleosidic monomers,e.g. as disclosed in Telenius et al, Genomics, 13: 718-725 (1992); Welshet al, Nucleic-Acids Research, 19: 5275-5279 (1991); Grothues et al,Nucleic Acids Research, 21: 1321-1322 (1993); Hartley, European patentapplication 90304496.4; Lam et al, Nature, 354: 82-84 (1991); Zuckermanet al, Int. J. Pept. Protein Research, 40: 498-507 (1992); and the like.Generally, these techniques simply call for the application of mixturesof the activated monomers to the growing oligonucleotide during thecoupling steps. Preferably, oligonucleotide tags and tag complements aresynthesized on a DNA synthesizer having a number of synthesis chamberswhich is greater than or equal to the number of different kinds of wordsused in the construction of the tags. That is, preferably there is asynthesis chamber corresponding to each type of word. In thisembodiment, words are added nucleotide-by-nucleotide, such that if aword consists of five nucleotides there are five monomer couplings ineach synthesis chamber. After a word is completely synthesized, thesynthesis supports are removed from the chambers, mixed, andredistributed back to the chambers for the next cycle of word addition.This latter embodiment takes advantage of the high coupling yields ofmonomer addition, e.g. in phosphoramidite chemistries.

Double stranded forms of tags may be made by separately synthesizing thecomplementary strands followed by mixing under conditions that permitduplex formation. Alternatively, double stranded tags may be formed byfirst synthesizing a single stranded repertoire linked to a knownoligonucleotide sequence that serves as a primer binding site. Thesecond strand is then synthesized by combining the single strandedrepertoire with a primer and extending with a polymerase. This latterapproach is described in Oliphant et al, Gene, 44: 177-183 (1986). Suchduplex tags may then be inserted into cloning vectors along with targetpolynucleotides for sorting and manipulating the target polynucleotidein accordance with the invention.

When tag complements are employed that are made up of nucleotides thathave enhanced binding characteristics, such as PNAs or oligonucleotideN3'→P5' phosphoramidates, sorting can be implemented through theformation of D-loops between tags comprising natural nucleotides andtheir PNA or phosphoramidate complements, as an alternative to the"stripping" reaction employing the 3'→5' exonuclease activity of a DNApolymerase to render a tag single stranded.

Oligonucleotide tags of the invention may range in length from 12 to 60,12 to 30, or 15 to 24 nucleotides or basepairs. Preferably,oligonucleotide tags range in length from 18 to 40 nucleotides orbasepairs. More preferably, oligonucleotide tags range in length from 25to 40 nucleotides or basepairs. In terms of preferred and more preferrednumbers of subunits, these ranges may be expressed as follows:

                  TABLE IV                                                        ______________________________________                                        Numbers of Subunits in Tags in Preferred Embodiments                          Monomers                                                                              Nucleotides in Oligonucleotide Tag                                    in Subunit                                                                            (12-60)     (18-40)      (25-40)                                      ______________________________________                                        3       4-20 subunits                                                                             6-13 subunits                                                                              8-13 subunits                                4       3-15 subunits                                                                             4-10 subunits                                                                              6-10 subunits                                5       2-12 subunits                                                                              3-8 subunits                                                                               5-8 subunits                                6       2-10 subunits                                                                              3-6 subunits                                                                               4-6 subunits                                ______________________________________                                    

Most preferably, oligonucleotide tags are single stranded and specifichybridization occurs via Watson-Crick pairing with a tag complement.

Preferably, repertoires of single stranded oligonucleotide tags of theinvention contain at least 100 members; more preferably, repertoires ofsuch tags contain at least 1000 members; and most preferably,repertoires of such tags contain at least 10,000 members.

Triplex Tags

In embodiments where specific hybridization occurs via triplexformation, coding of tag sequences follows the same principles as forduplex-forming tags; however, there are further constraints on theselection of subunit sequences. Generally, third strand association viaHoogsteen type of binding is most stable along homopyrimidine-homopurinetracks in a double stranded target. Usually, base triplets form in T-A*Tor C-G*C motifs (where "-" indicates Watson-Crick pairing and "*"indicates Hoogsteen type of binding); however, other motifs are alsopossible. For example, Hoogsteen base pairing permits parallel andantiparallel orientations between the third strand (the Hoogsteenstrand) and the purine-rich strand of the duplex to which the thirdstrand binds, depending on conditions and the composition of thestrands. There is extensive guidance in the literature for selectingappropriate sequences, orientation, conditions, nucleoside type (e.g.whether ribose or deoxyribose nucleosides are employed), basemodifications (e.g. methylated cytosine, and the like) in order tomaximize, or otherwise regulate, triplex stability as desired inparticular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci., 88:9397-9401 (1991); Roberts et al, Science, 258: 1463-1466 (1992); Robertset al, Proc. Natl. Acad. Sci., 93: 4320-4325 (1996); Distefano et al,Proc. Natl. Acad. Sci., 90: 1179-1183 (1993); Mergny et al,Biochemistry, 30: 9791-9798 (1991); Cheng et al, J. Am. Chem. Soc., 114:4465-4474 (1992); Beal and Dervan, Nucleic Acids Research, 20: 2773-2776(1992); Beal and Dervan, J. Am. Chem. Soc., 114: 4976-4982 (1992);Giovannangeli et al, Proc. Natl. Acad. Sci., 89: 8631-8635 (1992); Moserand Dervan, Science, 238: 645-650 (1987); McShan et al, J. Biol. Chem.,267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci., 89: 3840-3844(1992); Blume et al, Nucleic Acids Research, 20: 1777-1784 (1992);Thuong and Helene, Angew. Chem. Int. Ed. Engl. 32: 666-690 (1993);Escude et al, Proc. Natl. Acad. Sci., 93: 4365-4369 (1996); and thelike. Conditions for annealing single-stranded or duplex tags to theirsingle-stranded or duplex complements are well known, e.g. Ji et al,Anal. Chem. 65: 1323-1328 (1993); Cantor et al, U.S. Pat. No. 5,482,836;and the like. Use of triplex tags has the advantage of not requiring a"stripping" reaction with polymerase to expose the tag for annealing toits complement.

Preferably, oligonucleotide tags of the invention employing triplexhybridization are double stranded DNA and the corresponding tagcomplements are single stranded. More preferably, 5-methylcytosine isused in place of cytosine in the tag complements in order to broaden therange of pH stability of the triplex formed between a tag and itscomplement. Preferred conditions for forming triplexes are fullydisclosed in the above references. Briefly, hybridization takes place inconcentrated salt solution, e.g. 1.0 M NaCl, 1.0 M potassium acetate, orthe like, at pH below 5.5 (or 6.5 if 5-methylcytosine is employed).Hybridization temperature depends on the length and composition of thetag; however, for an 18-20-mer tag of longer, hybridization at roomtemperature is adequate. Washes may be conducted with less concentratedsalt solutions, e.g. 10 mM sodium acetate, 100 mM MgCl₂, pH 5.8, at roomtemperature. Tags may be eluted from their tag complements by incubationin a similar salt solution at pH 9.0.

Minimally cross-hybridizing sets of oligonucleotide tags that formtriplexes may be generated by the computer program of Appendix Ic, orsimilar programs. An exemplary set of double stranded 8-mer words arelisted below in capital letters with the corresponding complements insmall letters. Each such word differs from each of the other words inthe set by three base pairs.

                  TABLE V                                                         ______________________________________                                        Exemplary Minimally Cross-Hybridizing                                         Set of Double Stranded 8-mer Tags                                             ______________________________________                                        5'-AAGGAGAG                                                                              5'-AAAGGGGA                                                                               5'-AGAGAAGA                                                                               5'-AGGGGGGG                                3'-TTCCTCTC                                                                              3'-TTTCCCCT                                                                               3'-TCTCTTCT                                                                               3'-TCCCCCCC                                3'-ttcctctc                                                                              3'-tttcccct                                                                               3'-tctcttct                                                                               3'-tccccccc                                5'-AAAAAAAA                                                                              5'-AAGAGAGA                                                                               5'-AGGAAAAG                                                                               5'-GAAAGGAG                                3'-TTTTTTTT                                                                              3'-TTCTCTCT                                                                               3'-TCCTTTTC                                                                               3'-CTTTCCTC                                3'-tttttttt                                                                              3'-ttctctct                                                                               3'-tccttttc                                                                               3'-ctttcctc                                5'-AAAAAGGG                                                                              5'-AGAAGAGG                                                                               5'-AGGAAGGA                                                                               5'-GAAGAAGG                                3'-TTTTTCCC                                                                              3'-TCTTCTCC                                                                               3'-TCCTTCCT                                                                               3'-CTTCTTCC                                3'-tttttccc                                                                              3'-tcttctcc                                                                               3'-tccttcct                                                                               3'-cttcttcc                                5'-AAAGGAAG                                                                              5'-AGAAGGAA                                                                               5'-AGGGGAAA                                                                               5'-GAAGAGAA                                3'-TTTCCTTC                                                                              3'-TCTTCCTT                                                                               3'-TCCCCTTT                                                                               3'-CTTCTCTT                                3'-tttccttc                                                                              3'-tcttcctt                                                                               3'-tccccttt                                                                               3'-cttctctt                                ______________________________________                                    

                                      TABLE VI                                    __________________________________________________________________________    Repertoire Size of Various Double Stranded Tags                               That Form Triplexes with Their Tag Complements                                        Nucleotide                                                                    Difference                                                                    between  Maximal Size                                                         Oligonucleotides                                                                       or Minimally                                                                         Size of                                               Oligonucleotide                                                                       of Minimally                                                                           Cross- Repertoire                                                                          Size of                                         Word    Cross-   Hybridizing                                                                          with Four                                                                           Repertoire with                                 Length  Hybridizing Set                                                                        Set    Words Five Words                                      __________________________________________________________________________     4      2         8     4096  3.2 × 10.sup.4                             6      3         8     4096  3.2 × 10.sup.4                             8      3        16     6.5 × 10.sup.4                                                                1.05 × 10.sup.6                           10      5         8     4096                                                  15      5        92                                                           20      6        765                                                          20      8        92                                                           20      10       22                                                           __________________________________________________________________________

Preferably, repertoires of double stranded oligonucleotide tags of theinvention contain at least 10 members; more preferably, repertoires ofsuch tags contain at least 100 members. Preferably, words are between 4and 8 nucleotides in length for combinatorially synthesized doublestranded oligonucleotide tags, and oligonucleotide tags are between 12and 60, 12 to 30, or 15 to 24 base pairs in length. More preferably,such tags are between 18 and 40 base pairs in length.

Solid Phase Supports

Solid phase supports for use with the invention may have a wide varietyof forms, including microparticles, beads, and membranes, slides,plates, micromachined chips, and the like. Likewise, solid phasesupports of the invention may comprise a wide variety of compositions,including glass, plastic, silicon, alkanethiolate-derivatized gold,cellulose, low cross-linked and high cross-linked polystyrene, silicagel, polyamide, and the like. Preferably, either a population ofdiscrete particles are employed such that each has a uniform coating, orpopulation, of complementary sequences of the same tag (and no other),or a single or a few supports are employed with spatially discreteregions each containing a uniform coating, or population, ofcomplementary sequences to the same tag (and no other). In the latterembodiment, the area of the regions may vary according to particularapplications; usually, the regions range in area from several μm², e.g.3-5, to several hundred μm², e.g. 100-500 or e.g. 10 to 1000Micrometers². Preferably, such regions are spatially discrete so thatsignals generated by events, e.g. fluorescent emissions, at adjacentregions can be resolved by the detection system being employed. In someapplications, it may be desirable to have regions with uniform coatingsof more than one tag complement, e.g. for simultaneous sequenceanalysis, or for bringing separately tagged molecules into closeproximity.

Tag complements may be used with the solid phase support that they aresynthesized on, or they may be separately synthesized and attached to asolid phase support for use, e.g. as disclosed by Lund et al, NucleicAcids Research, 16: 10861-10880 (1988); Albretsen et al, Anal. Biochem.,189: 40-50 (1990); Wolf et al, Nucleic Acids Research, 15: 2911-2926(1987); or Ghosh et al, Nucleic Acids Research, 15: 5353-5372 (1987).Preferably, tag complements are synthesized on and used with the samesolid phase support, which may comprise a variety of forms and include avariety of linking moieties. Such supports may comprise microparticlesor arrays, or matrices, of regions where uniform populations of tagcomplements are synthesized. A wide variety of microparticle supportsmay be used with the invention, including microparticles made ofcontrolled pore glass (CPG), highly cross-linked polystyrene, acryliccopolymers, cellulose, nylon, dextran, latex, polyacrolein, and thelike, disclosed in the following exemplary references: Meth. Enzymol.,Section A, pages 11-147, vol. 44 (Academic Press, New York, 1976); U.S.Pat. Nos. 4,678,814; 4,413,070; and 4,046;720; and Pon, Chapter 19, inAgrawal, editor, Methods in Molecular Biology, Vol. 20, (Humana Press,Totowa, N.J., 1993). Microparticle supports further include commerciallyavailable nucleoside-derivatized CPG and polystyrene beads (e.g.available from Applied Biosystems, Foster City, Calif.); derivatizedmagnetic beads; polystyrene grafted with polyethylene glycol (e.g.,TentaGel™, Rapp Polymere, Tubingen Germany); and the like. Selection ofthe support characteristics, such as material, porosity, size, shape,and the like, and the type of linking moiety employed depends on theconditions under which the tags are used. For example, in applicationsinvolving successive processing with enzymes, supports and linkers thatminimize steric hindrance of the enzymes and that facilitate access tosubstrate are preferred. Other important factors to be considered inselecting the most appropriate microparticle support include sizeuniformity, efficiency as a synthesis support, degree to which surfacearea known, and optical properties, e.g. as explain more fully below,clear smooth beads provide instrumentational advantages when handlinglarge numbers of beads on a surface.

Exemplary linking moieties for attaching and/or synthesizing tags onmicroparticle surfaces are disclosed in Pon et al, Biotechniques,6:768-775 (1988); Webb, U.S. Pat. No. 4,659,774; Barany et al,International patent application PCT/US91/06103; Brown et al, J. Chem.Soc. Commun., 1989: 891-893; Damha et al, Nucleic Acids Research, 18:3813-3821 (1990); Beattie et al, Clinical Chemistry, 39: 719-722 (1993);Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992); andthe like.

As mentioned above, tag complements may also be synthesized on a single(or a few) solid phase support to form an array of regions uniformlycoated with tag complements. That is, within each region in such anarray the same tag complement is synthesized. Techniques forsynthesizing such arrays are disclosed in McGall et al, Internationalapplication PCT/US93/03767; Pease et al, Proc. Natl. Acad. Sci., 91:5022-5026 (1994); Southern and Maskos, International applicationPCT/GB89/01114; Maskos and Southern (cited above); Southern et al,Genomics, 13: 1008-1017 (1992); and Maskos and Southern, Nucleic AcidsResearch, 21: 4663-4669 (1993).

Preferably, the invention is implemented with microparticles or beadsuniformly coated with complements of the same tag sequence.Microparticle supports and methods of covalently or noncovalentlylinking oligonucleotides to their surfaces are well known, asexemplified by the following references: Beaucage and Iyer (citedabove); Gait, editor, Oligonucleotide Synthesis: A Practical Approach(IRL Press, Oxford, 1984); and the references cited above. Generally,the size and shape of a microparticle is not critical; however,microparticles in the size range of a few, e.g. 1-2, to several hundred,e.g. 200-1000 μm diameter are preferable, as they facilitate theconstruction and manipulation of large repertoires of oligonucleotidetags with minimal reagent and sample usage.

In some preferred applications, commercially available controlled-poreglass (CPG) or polystyrene supports are employed as solid phase supportsin the invention. Such supports come available with base-labile linkersand initial nucleosides attached, e.g. Applied Biosystems (Foster City,Calif.). Preferably, microparticles having pore size between 500 and1000 angstroms are employed.

In other preferred applications, non-porous microparticles are employedfor their optical properties, which may be advantageously used whentracking large numbers of microparticles on planar supports, such as amicroscope slide. Particularly preferred non-porous microparticles arethe glycidal methacrylate (GMA) beads available from Bangs Laboratories(Carmel, Ind.). Such microparticles are useful in a variety of sizes andderivatized with a variety of linkage groups for synthesizing tags ortag complements. Preferably, for massively parallel manipulations oftagged microparticles, 5 μm diameter GMA beads are employed.

Attaching Tags to Polynucleotides For Sorting onto Solid Phase Supports

An important aspect of the invention is the sorting and attachment of apopulations of polynucleotides, e.g. from a cDNA library, tomicroparticles or to separate regions on a solid phase support such thateach microparticle or region has substantially only one kind ofpolynucleotide attached. This objective is accomplished by insuring thatsubstantially all different polynucleotides have different tagsattached. This condition, in turn, is brought about by taking a sampleof the full ensemble of tag-polynucleotide conjugates for analysis. (Itis acceptable that identical polynucleotides have different tags, as itmerely results in the same polynucleotide being operated on or analyzedtwice in two different locations.) Such sampling can be carried outeither overtly--for example, by taking a small volume from a largermixture--after the tags have been attached to the polynucleotides, itcan be carried out inherently as a secondary effect of the techniquesused to process the polynucleotides and tags, or sampling can be carriedout both overtly and as an inherent part of processing steps.

Preferably, in constructing a cDNA library where substantially alldifferent cDNAs have different tags, a tag repertoire is employed whosecomplexity, or number of distinct tags, greatly exceeds the total numberof mRNAs extracted from a cell or tissue sample. Preferably, thecomplexity of the tag repertoire is at least 10 times that of thepolynucleotide population; and more preferably, the complexity of thetag repertoire is at least 100 times that of the polynucleotidepopulation. Below, a protocol is disclosed for cDNA library constructionusing a primer mixture that contains a full repertoire of exemplary9-word tags. Such a mixture of tag-containing primers has a complexityof 8⁹, or about 1.34×10⁸. As indicated by Winslow et al, Nucleic AcidsResearch, 19: 3251-3253 (1991), mRNA for library construction can beextracted from as few as 10-100 mammalian cells. Since a singlemammalian cell contains about 5×10⁵ copies of mRNA molecules of about3.4×10⁴ different kinds, by standard techniques one can isolate the mRNAfrom about 100 cells, or (theoretically) about 5×10⁷ mRNA molecules.Comparing this number to the complexity of the primer mixture shows thatwithout any additional steps, and even assuming that mRNAs are convertedinto cDNAs with perfect efficiency (1% efficiency or less is moreaccurate), the cDNA library construction protocol results in apopulation containing no more than 37% of the total number of differenttags. That is, without any overt sampling step at all, the protocolinherently generates a sample that comprises 37%, or less, of the tagrepertoire. The probability of obtaining a double under these conditionsis about 5%, which is within the preferred range. With mRNA from 10cells, the fraction of the tag repertoire sampled is reduced to only3.7%, even assuming that all the processing steps take place at 100%efficiency. In fact, the efficiencies of the processing steps forconstructing cDNA libraries are very low, a "rule of thumb" being thatgood library should contain about 10⁸ cDNA clones from mRNA extractedfrom 10⁶ mammalian cells.

Use of larger amounts of mRNA in the above protocol, or for largeramounts of polynucleotides in general, where the number of suchmolecules exceeds the complexity of the tag repertoire, atag-polynucleotide conjugate mixture potentially contains every possiblepairing of tags and types of mRNA or polynucleotide. In such cases,overt sampling may be implemented by removing a sample volume after aserial dilution of the starting mixture of tag-polynucleotideconjugates. The amount of dilution required depends on the amount ofstarting material and the efficiencies of the processing steps, whichare readily estimated.

If mRNA were extracted from 10⁶ cells (which would correspond to about0.5 μg of poly(A)⁺ RNA), and if primers were present in about 10-100fold concentration excess--as is called for in a typical protocol, e.g.Sambrook et al, Molecular Cloning, Second Edition, page 8.61 [10 μL 1.8kb mRNA at 1 mg/mL equals about 1.68×10⁻¹¹ moles and 10 μL 18-mer primerat 1 mg/mL equals about 1.68×10⁻⁹ moles], then the total number oftag-polynucleotide conjugates in a cDNA library would simply be equal toor less than the starting number of mRNAs, or about 5×10¹¹ vectorscontaining tag-polynucleotide conjugates--again this assumes that eachstep in cDNA construction--first strand synthesis, second strandsynthesis, ligation into a vector--occurs with perfect efficiency, whichis a very conservative estimate. The actual number is significantlyless.

If a sample of n tag-polynucleotide conjugates are randomly drawn from areaction mixture--as could be effected by taking a sample volume, theprobability of drawing conjugates having the same tag is described bythe Poisson distribution, P(r)=e⁻λ (λ)^(r) /r, where r is the number ofconjugates having the same tag and λ=np, where p is the probability of agiven tag being selected. If n=10⁶ and p=1/(1.34×10⁸), then λ=0.00746and P(2)=2.76×10⁻⁵. Thus, a sample of one million molecules gives riseto an expected number of doubles well within the preferred range. Such asample is readily obtained as follows: Assume that the 5×10¹¹ mRNAs areperfectly converted into 5×10¹¹ vectors with tag-cDNA conjugates asinserts and that the 5×10¹¹ vectors are in a reaction solution having avolume of 100 μl. Four 10-fold serial dilutions may be carried out bytransferring 10 μl from the original solution into a vessel containing90 μl of an appropriate buffer, such as TE. This process may be repeatedfor three additional dilutions to obtain a 100 μl solution containing5×10⁵ vector molecules per μl. A 2 μl aliquot from this solution yields10⁶ vectors containing tag-cDNA conjugates as inserts. This sample isthen amplified by straight forward transformation of a competent hostcell followed by culturing.

Of course, as mentioned above, no step in the above process proceedswith perfect efficiency. In particular, when vectors are employed toamplify a sample of tag-polynucleotide conjugates, the step oftransforming a host is very inefficient. Usually, no more than 1% of thevectors are taken up by the host and replicated. Thus, for such a methodof amplification, even fewer dilutions would be required to obtain asample of 10⁶ conjugates.

A repertoire of oligonucleotide tags can be conjugated to a populationof polynucleotides in a number of ways, including direct enzymaticligation, amplification, e.g. via PCR, using primers containing the tagsequences, and the like. The initial ligating step produces a very largepopulation of tag-polynucleotide conjugates such that a single tag isgenerally attached to many different polynucleotides. However, as notedabove, by taking a sufficiently small sample of the conjugates, theprobability of obtaining "doubles," i.e. the same tag on two differentpolynucleotides, can be made negligible. Generally, the larger thesample the greater the probability of obtaining a double. Thus, a designtrade-off exists between selecting a large sample of tag-polynucleotideconjugates--which, for example, ensures adequate coverage of a targetpolynucleotide in a shotgun sequencing operation or adequaterepresentation of a rapidly changing mRNA pool, and selecting a smallsample which ensures that a minimal number of doubles will be present.In most embodiments, the presence of doubles merely adds an additionalsource of noise or, in the case of sequencing, a minor complication inscanning and signal processing, as microparticles giving multiplefluorescent signals can simply be ignored.

As used herein, the term "substantially all" in reference to attachingtags to molecules, especially polynucleotides, is meant to reflect thestatistical nature of the sampling procedure employed to obtain apopulation of tag-molecule conjugates essentially free of doubles. Themeaning of substantially all in terms of actual percentages oftag-molecule conjugates depends on how the tags are being employed.Preferably, for nucleic acid sequencing, substantially all means that atleast eighty percent of the polynucleotides have unique tags attached.More preferably, it means that at least ninety percent of thepolynucleotides have unique tags attached. Still more preferably, itmeans that at least ninety-five percent of the polynucleotides haveunique tags attached. And, most preferably, it means that at leastninety-nine percent of the polynucleotides have unique tags attached.

Preferably, when the population of polynucleotides consists of messengerRNA (mRNA), oligonucleotides tags may be attached by reversetranscribing the mRNA with a set of primers preferably containingcomplements of tag sequences. An exemplary set of such primers couldhave the following sequence: (SEQ ID NO:1)5'-mRNA- [A]_(n) -3' [T]₁₉GG[W,W,W,C]₉ ACCAGCTGATC-5'-biotin

where "[W,W,W,C]₉ " represents the sequence of an oligonucleotide tag ofnine subunits of four nucleotides each and "[W,W,W,C]" represents thesubunit sequences listed above, i.e. "W" represents T or A. Theunderlined sequences identify an optional restriction endonuclease sitethat can be used to release the polynucleotide from attachment to asolid phase support via the biotin, if one is employed. For the aboveprimer, the complement attached to a microparticle could have the form:(SEQ ID NO:2)

    5'-[G,W,W,W].sub.9 TGG-linker-microparticle

After reverse transcription, the mRNA is removed, e.g. by RNase Hdigestion, and the second strand of the cDNA is synthesized using, forexample, a primer of the following form: (SEQ ID NO:3)

    5'-NRRGATCYNNN-3'

where N is any one of A, T, G, or C; R is a purine-containingnucleotide, and Y is a pyrimidine-containing nucleotide. This particularprimer creates a Bst Y1 restriction site in the resulting doublestranded DNA which, together with the Sal I site, facilitates cloninginto a vector with, for example, Bam HI and Xho I sites. After Bst Y1and Sal I digestion, the exemplary conjugate would have the form: (SEQID NO:4)5'-RCGACCA[C,W,W,W]₉ GG[T]₁₉ - cDNA -NNNR GGT[G,W,W,W]₉CC[A]₁₉ - rDNA -NNNYCTAG-5'

The polynucleotide-tag conjugates may then be manipulated using standardmolecular biology techniques. For example, the above conjugate--which isactually a mixture--may be inserted into commercially available cloningvectors, e.g. Stratagene Cloning System (La Jolla, Calif.); transfectedinto a host, such as a commercially available host bacteria; which isthen cultured to increase the number of conjugates. The cloning vectorsmay then be isolated using standard techniques, e.g. Sambrook et al,Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989). Alternatively, appropriate adaptors and primers may beemployed so that the conjugate population can be increased by PCR.

Preferably, when the ligase-based method of sequencing is employed, theBst Y1 and Sal I digested fragments are cloned into a Bam HI-/XhoI-digested vector having the following single-copy restriction sites:(SEQ ID NO:5)5'-GAGGATGCCTTTATGGATCCACTCGAGATCCCAATCCA-3' FokI BamHIXhoI

This adds the Fok I site which will allow initiation of the sequencingprocess discussed more fully below.

Tags can be conjugated to cDNAs of existing libraries by standardcloning methods. cDNAs are excised from their existing vector, isolated,and then ligated into a vector containing a repertoire of tags.Preferably, the tag-containing vector is linearized by cleaving with tworestriction enzymes so that the excised cDNAs can be ligated in apredetermined orientation. The concentration of the linearizedtag-containing vector is in substantial excess over that of the cDNAinserts so that ligation provides an inherent sampling of tags.

A general method for exposing the single stranded tag afteramplification involves digesting a target polynucleotide-containingconjugate with the 3'→3' exonuclease activity of T4 DNA polymerase, or alike enzyme. When used in the presence of a single deoxynucleosidetriphosphate, such a polymerase will cleave nucleotides from 3' recessedends present on the non-template strand of a double stranded fragmentuntil a complement of the single deoxynucleoside triphosphate is reachedon the template strand. When such a nucleotide is reached the 3'→5'digestion effectively ceases, as the polymerase's extension activityadds nucleotides at a higher rate than the excision activity removesnucleotides. Consequently, single stranded tags constructed with threenucleotides are readily prepared for loading onto solid phase supports.

The technique may also be used to preferentially methylate interior FokI sites of a target polynucleotide while leaving a single Fok I site atthe terminus of the polynucleotide unmethylated. First, the terminal FokI site is rendered single stranded using a polymerase with deoxycytidinetriphosphate. The double stranded portion of the fragment is thenmethylated, after which the single stranded terminus is filled in with aDNA polymerase in the presence of all four nucleoside triphosphates,thereby regenerating the Fok I site. Clearly, this procedure can begeneralized to endonucleases other than Fok I.

After the oligonucleotide tags are prepared for specific hybridization,e.g. by rendering them single stranded as described above, thepolynucleotides are mixed with microparticles containing thecomplementary sequences of the tags under conditions that favor theformation of perfectly matched duplexes between the tags and theircomplements. There is extensive guidance in the literature for creatingthese conditions. Exemplary references providing such guidance includeWetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991); Sambrook et al, Molecular Cloning: A Laboratory Manual,2nd Edition (Cold Spring Harbor Laboratory, New York, 1989); and thelike. Preferably, the hybridization conditions are sufficientlystringent so that only perfectly matched sequences form stable duplexes.Under such conditions the polynucleotides specifically hybridizedthrough their tags may be ligated to the complementary sequencesattached to the microparticles. Finally, the microparticles are washedto remove polynucleotides with unligated and/or mismatched tags.

When CPG microparticles conventionally employed as synthesis supportsare used, the density of tag complements on the microparticle surface istypically greater than that necessary for some sequencing operations.That is, in sequencing approaches that require successive treatment ofthe attached polynucleotides with a variety of enzymes, densely spacedpolynucleotides may tend to inhibit access of the relatively bulkyenzymes to the polynucleotides. In such cases, the polynucleotides arepreferably mixed with the microparticles so that tag complements arepresent in significant excess, e.g. from 10:1 to 100:1, or greater, overthe polynucleotides. This ensures that the density of polynucleotides onthe microparticle surface will not be so high as to inhibit enzymeaccess. Preferably, the average inter-polynucleotide spacing on themicroparticle surface is on the order of 30-100 nm. Guidance inselecting ratios for standard CPG supports and Ballotini beads (a typeof solid glass support) is found in Maskos and Southern, Nucleic AcidsResearch, 20: 1679-1684 (1992). Preferably, for sequencing applications,standard CPG beads of diameter in the range of 20-50 μm are loaded withabout 10⁵ polynucleotides, and GMA beads of diameter in the range of5-10 μm are loaded with a few tens of thousand polynucleotide, e.g.4×10⁴ to 6×10⁴.

In the preferred embodiment, tag complements are synthesized onmicroparticles combinatorially; thus, at the end of the synthesis, oneobtains a complex mixture of microparticles from which a sample is takenfor loading tagged polynucleotides. The size of the sample ofmicroparticles will depend on several factors, including the size of therepertoire of tag complements, the nature of the apparatus for used forobserving loaded microparticles--e.g. its capacity, the tolerance formultiple copies of microparticles with the same tag complement (i.e."bead doubles"), and the like. The following table provide guidanceregarding microparticle sample size, microparticle diameter, and theapproximate physical dimensions of a packed array of microparticles ofvarious diameters.

    ______________________________________                                        Microparticle diameter                                                                    5 μm   10 μm 20 μm                                                                             40 μm                                Max. no.              3 × 10.sup.5                                                                     1.26 × 10.sup.6                                                                5 × 10.sup.6                      polynucleotides loaded                                                        at 1 per 10.sup.5 sq.                                                         angstrom                                                                      Approx. area of                                                                           .45 × .45 cm                                                                      1 × 1 cm                                                                         2× 2 cm                                                                        4 × 4 cm                          monolayer of 10.sup.6                                                         microparticles                                                                ______________________________________                                    

The probability that the sample of microparticles contains a given tagcomplement or is present in multiple copies is described by the Poissondistribution, as indicated in the following table.

                  TABLE VII                                                       ______________________________________                                                                        Fraction of                                                                   microparticles in                             Number of                                                                             Fraction of             sample carrying                               micro-  repertoire Fraction of  same tag                                      particles                                                                             of tag     microparticles in                                                                          complement as one                             in sample (as                                                                         complements                                                                              sample with unique                                                                         other microparticle                           fraction of                                                                           present    tag complement                                                                             in sample                                     repertoire                                                                            in sample, attached,    ("bead doubles"),                             size), m                                                                              1 - e.sup.-m                                                                             m(e.sup.-m)/2                                                                              m.sup.2 (e.sup.-m)/2                          ______________________________________                                        1.000   0.63       0.37         0.18                                          .693    0.50       0.35         0.12                                          .405    0.33       0.27         0.05                                          .285    0.25       0.21         0.03                                          .223    0.20       0.18         0.02                                          .105    0.10       0.09         0.005                                         .010    0.01       0.01                                                       ______________________________________                                    

High Specificity Sorting and Panning

The kinetics of sorting depends on the rate of hybridization ofoligonucleotide tags to their tag complements which, in turn, depends onthe complexity of the tags in the hybridization reaction. Thus, a tradeoff exists between sorting rate and tag complexity, such that anincrease in sorting rate may be achieved at the cost of reducing thecomplexity of the tags involved in the hybridization reaction. Asexplained below, the effects of this trade off may be ameliorated by"panning."

Specificity of the hybridizations may be increased by taking asufficiently small sample so that both a high percentage of tags in thesample are unique and the nearest neighbors of substantially all thetags in a sample differ by at least two words. This latter condition maybe met by taking a sample that contains a number of tag-polynucleotideconjugates that is about 0.1 percent or less of the size of therepertoire being employed. For example, if tags are constructed witheight words selected from Table II, a repertoire of 8⁸, or about1.67×10⁷, tags and tag complements are produced. In a library oftag-cDNA conjugates as described above, a 0.1 percent sample means thatabout 16,700 different tags are present. If this were loaded directlyonto a repertoire-equivalent of microparticles, or in this example asample of 1.67×10⁷ microparticles, then only a sparse subset of thesampled microparticles would be loaded. The density of loadedmicroparticles can be increase--for example, for more efficientsequencing--by undertaking a "panning" step in which the sampledtag-cDNA conjugates are used to separate loaded microparticles fromunloaded microparticles. Thus, in the example above, even though a "0.1percent" sample contains only 16,700 cDNAs, the sampling and panningsteps may be repeated until as many loaded microparticles as desired areaccumulated.

A panning step may be implemented by providing a sample of tag-cDNAconjugates each of which contains a capture moiety at an end opposite,or distal to, the oligonucleotide tag. Preferably, the capture moiety isof a type which can be released from the tag-cDNA conjugates, so thatthe tag-cDNA conjugates can be sequenced with a single-base sequencingmethod. Such moieties may comprise biotin, digoxigenin, or like ligands,a triplex binding region, or the like. Preferably, such a capture moietycomprises a biotin component. Biotin may be attached to tag-cDNAconjugates by a number of standard techniques. If appropriate adapterscontaining PCR primer binding sites are attached to tag-cDNA conjugates,biotin may be attached by using a biotinylated primer in anamplification after sampling. Alternatively, if the tag-cDNA conjugatesare inserts of cloning vectors, biotin may be attached after excisingthe tag-cDNA conjugates by digestion with an appropriate restrictionenzyme followed by isolation and filling in a protruding strand distalto the tags with a DNA polymerase in the presence of biotinylateduridine triphosphate.

After a tag-cDNA conjugate is captured, it may be released from thebiotin moiety in a number of ways, such as by a chemical linkage that iscleaved by reduction, e.g. Herman et al, Anal. Biochem., 156: 48-55(1986), or that is cleaved photochemically, e.g. Olejnik et al, NucleicAcids Research, 24: 361-366 (1996), or that is cleaved enzymatically byintroducing a restriction site in the PCR primer. The latter embodimentcan be exemplified by considering the library of tag-polynucleotideconjugates described above: (SEQ ID NO:4)5'-RCGACCA[C,W,W,W]₉ GG[T]₁₉ -cDNA -NNNR GGT[G,W,W,W]₉ CC[A]₁₉ - rDNA -NNNYCTAG-5'

The following adapters may be ligated to the ends of these fragments topermit amplification by PCR:5'-XXXXXXXXXXXXXXXXXXXX (SEQ ID NO:6)XXXXXXXXXXXXXXXXXXXXYGAT Right AdapterGATCZZACTAGTZZZZZZZZZZZZ-3' (SEQID NO:7) ZZGATCAZZZZZZZZZZZZZ Left AdapterZZTGATCAZZZZZZZZZZZZ-5'-biotin(SEQ ID NO:8) Left Primer

where "ACTAGT" is a Spe I recognition site (which leaves a staggeredcleavage ready for single base sequencing), and the X's and Z's arenucleotides selected so that the annealing and dissociation temperaturesof the respective primers are approximately the same. After ligation ofthe adapters and amplification by PCR using the biotinylated primer, thetags of the conjugates are rendered single stranded by the exonucleaseactivity of T4 DNA polymerase and conjugates are combined with a sampleof microparticles, e.g. a repertoire equivalent, with tag complementsattached. After annealing under stringent conditions (to minimizemis-attachment of tags), the conjugates are preferably ligated to theirtag complements and the loaded microparticles are separated from theunloaded microparticles by capture with avidinated magnetic beads, orlike capture technique.

Returning to the example, this process results in the accumulation ofabout 10,500 (=16,700×0.63) loaded microparticles with different tags,which may be released from the magnetic beads by cleavage with Spe I. Byrepeating this process 40-50 times with new samples of microparticlesand tag-cDNA conjugates, 4-5×10⁵ cDNAs can be accumulated by pooling thereleased microparticles. The pooled microparticles may then besimultaneously sequenced by a single-base sequencing technique.

Determining how many times to repeat the sampling and panning steps--ormore generally, determining how many cDNAs to analyze, depends on one'sobjective. If the objective is to monitor the changes in abundance ofrelatively common sequences, e.g. making up 5% or more of a population,then relatively small samples, i.e. a small fraction of the totalpopulation size, may allow statistically significant estimates ofrelative abundances. On the other hand, if one seeks to monitor theabundances of rare sequences, e.g. making up 0.1% or less of apopulation, then large samples are required. Generally, there is adirect relationship between sample size and the reliability of theestimates of relative abundances based on the sample. There is extensiveguidance in the literature on determining appropriate sample sizes formaking reliable statistical estimates, e.g. Koller et al, Nucleic AcidsResearch, 23:185-191 (1994); Good, Biometrika, 40: 16-264 (1953); Bungeet al, J. Am. Stat. Assoc., 88: 364-373 (1993); and the like.Preferably, for monitoring changes in gene expression based on theanalysis of a series of cDNA libraries containing 10⁵ to 10⁸ independentclones of 3.0-3.5×10⁴ different sequences, a sample of at least 10⁴sequences are accumulated for analysis of each library. More preferably,a sample of at least 10⁵ sequences are accumulated for the analysis ofeach library; and most preferably, a sample of at least 5×10⁵ sequencesare accumulated for the analysis of each library. Alternatively, thenumber of sequences sampled is preferably sufficient to estimate therelative abundance of a sequence present at a frequency within the rangeof 0.1% to 5% with a 95% confidence limit no larger than 0.1% of thepopulation size.

Single Base DNA Sequencing

The present invention can be employed with conventional methods of DNAsequencing, e.g. as disclosed by Hultman et al, Nucleic Acids Research,17: 4937-4946 (1989). However, for parallel, or simultaneous, sequencingof multiple polynucleotides, a DNA sequencing methodology is preferredthat requires neither electrophoretic separation of closely sized DNAfragments nor analysis of cleaved nucleotides by a separate analyticalprocedure, as in peptide sequencing. Preferably, the methodology permitsthe stepwise identification of nucleotides, usually one at a time, in asequence through successive cycles of treatment and detection. Suchmethodologies are referred to herein as "single base" sequencingmethods. Single base approaches are disclosed in the followingreferences: Cheeseman, U.S. Pat. No. 5,302,509; Tsien et al,International application WO 91/06678; Rosenthal et al, Internationalapplication WO 93/21340; Canard et al, Gene, 148: 1-6 (1994); andMetzker et al, Nucleic Acids Research, 22: 4259-4267 (1994).

A "single base" method of DNA sequencing which is suitable for use withthe present invention and which requires no electrophoretic separationof DNA fragments is described in International applicationPCT/US95/03678. Briefly, the method comprises the following steps: (a)ligating a probe to an end of the polynucleotide having a protrudingstrand to form a ligated complex, the probe having a complementaryprotruding strand to that of the polynucleotide and the probe having anuclease recognition site; (b) removing unligated probe from the ligatedcomplex; (c) identifying one or more nucleotides in the protrudingstrand of the polynucleotide by the identity of the ligated probe; (d)cleaving the ligated complex with a nuclease; and (e) repeating steps(a) through (d) until the nucleotide sequence of the polynucleotide, ora portion thereof, is determined.

A single signal generating moiety, such as a single fluorescent dye, maybe employed when sequencing several different target polynucleotidesattached to different spatially addressable solid phase supports, suchas fixed microparticles, in a parallel sequencing operation. This may beaccomplished by providing four sets of probes that are appliedsequentially to the plurality of target polynucleotides on the differentmicroparticles. An exemplary set of such probes are shown below:

    __________________________________________________________________________    Set 1    Set 2     Set 3    Set 4                                             __________________________________________________________________________    ANNNN . . . NN                                                                         dANNNN . . . NN                                                                         dANNNN . . . NN                                                                        dANNNN . . . NN                                   N . . . NNTT . . . T*                                                                  d N . . . NNTT . . . T                                                                  N . . . NNTT . . . T                                                                   N . . . NNTT . . . T                              dCNNNN . . . NN                                                                        CNNNN . . . NN                                                                          dCNNNN . . . NN                                                                        dCNNNN . . . NN                                   N . . . NNTT . . . T                                                                   N . . . NNTT . . . T*                                                                   N . . . NNTT . . . T                                                                   N . . . NNTT . . . T                              dGNNNN . . . NN                                                                        dGNNNN . . . NN                                                                         GNNNN . . . NN                                                                         dGNNNN . . . NN                                   N . . . NNTT . . . T                                                                   N . . . NNTT . . . T                                                                    N . . . NNTT . . . T*                                                                  N . . . NNTT . . . T                              dTNNNN . . . NN                                                                        dTNNNN . . . NN                                                                         dTNNNN . . . NN                                                                        TNNNN . . . NN                                    N . . . NNTT . . . T                                                                   N . . . NNTT . . . T                                                                    N . . . NNTT . . . T                                                                   N . . . NNTT . . . T*                             __________________________________________________________________________

where each of the listed probes represents a mixture of 4³ =64oligonucleotides such that the identity of the 3' terminal nucleotide ofthe top strand is fixed and the other positions in the protruding strandare filled by every 3-mer permutation of nucleotides, or complexityreducing analogs. The listed probes are also shown with a singlestranded poly-T tail with a signal generating moiety attached to theterminal thymidine, shown as "T*". The "d" on the unlabeled probesdesignates a ligation-blocking moiety or absense of 3'-hydroxyl, whichprevents unlabeled probes from being ligated. Preferably, such3'-terminal nucleotides are dideoxynucleotides. In this embodiment, theprobes of set 1 are first applied to the plurality of targetpolynucleotides and treated with a ligase so that target polynucleotideshaving a thymidine complementary to the 3' terminal adenosine of thelabeled probes are ligated. The unlabeled probes are simultaneouslyapplied to minimize inappropriate ligations. The locations of the targetpolynucleotides that form ligated complexes with probes terminating in"A" are identified by the signal generated by the label carried on theprobe. After washing and cleavage, the probes of set 2 are applied. Inthis case, target polynucleotides forming ligated complexes with probesterminating in "C" are identified by location. Similarly, the probes ofsets 3 and 4 are applied and locations of positive signals identified.This process of sequentially applying the four sets of probes continuesuntil the desired number of nucleotides are identified on the targetpolynucleotides. Clearly, one of ordinary skill could construct similarsets of probes that could have many variations, such as havingprotruding strands of different lengths, different moieties to blockligation of unlabeled probes, different means for labeling probes, andthe like.

Apparatus for Observing Enzymatic Processes and/or Binding Events atMicroparticle Surfaces

An objective of the invention is to sort identical molecules,particularly polynucleotides, onto the surfaces of microparticles by thespecific hybridization of tags and their complements. Once such sortinghas taken place, the presence of the molecules or operations performedon them can be detected in a number of ways depending on the nature ofthe tagged molecule, whether microparticles are detected separately orin "batches," whether repeated measurements are desired, and the like.Typically, the sorted molecules are exposed to ligands for binding, e.g.in drug development, or are subjected chemical or enzymatic processes,e.g. in polynucleotide sequencing. In both of these uses it is oftendesirable to simultaneously observe signals corresponding to such eventsor processes on large numbers of microparticles. Microparticles carryingsorted molecules (referred to herein as "loaded" microparticles) lendthemselves to such large scale parallel operations, e.g. as demonstratedby Lam et al (cited above).

Preferably, whenever light-generating signals, e.g. chemiluminescent,fluorescent, or the like, are employed to detect events or processes,loaded microparticles are spread on a planar substrate, e.g. a glassslide, for examination with a scanning system, such as described inInternational patent applications PCT/US91/09217, PCT/NL90/00081, andPCT/US95/01886. The scanning system should be able to reproducibly scanthe substrate and to define the positions of each microparticle in apredetermined region by way of a coordinate system. In polynucleotidesequencing applications, it is important that the positionalidentification of microparticles be repeatable in successive scan steps.

Such scanning systems may be constructed from commercially availablecomponents, e.g. x-y translation table controlled by a digital computerused with a detection system comprising one or more photomultipliertubes, or alternatively, a CCD array, and appropriate optics, e.g. forexciting, collecting, and sorting fluorescent signals. In someembodiments a confocal optical system may be desirable. An exemplaryscanning system suitable for use in four-color sequencing is illustrateddiagrammatically in FIG. 2. Substrate 300, e.g. a microscope slide withfixed microparticles, is placed on x-y translation table 302, which isconnected to and controlled by an appropriately programmed digitalcomputer 304 which may be any of a variety of commercially availablepersonal computers, e.g. 486-based machines or PowerPC model 7100 or8100 available form Apple Computer (Cupertino, Calif.). Computersoftware for table translation and data collection functions can beprovided by commercially available laboratory software, such as LabWindows, available from National Instruments.

Substrate 300 and table 302 are operationally associated with microscope306 having one or more objective lenses 308 which are capable ofcollecting and delivering light to microparticles fixed to substrate300. Excitation beam 310 from light source 312, which is preferably alaser, is directed to beam splitter 314, e.g. a dichroic mirror, whichre-directs the beam through microscope 306 and objective lens 308 which,in turn, focuses the beam onto substrate 300. Lens 308 collectsfluorescence 316 emitted from the microparticles and directs it throughbeam splitter 314 to signal distribution optics 318 which, in turn,directs fluorescence to one or more suitable opto-electronic devices forconverting some fluorescence characteristic, e.g. intensity, lifetime,or the like, to an electrical signal. Signal distribution optics 318 maycomprise a variety of components standard in the art, such as bandpassfilters, fiber optics, rotating mirrors, fixed position mirrors andlenses, diffraction gratings, and the like. As illustrated in FIG. 2,signal distribution optics 318 directs fluorescence 316 to four separatephotomultiplier tubes, 330, 332, 334, and 336, whose output is thendirected to pre-amps and photon counters 350, 352, 354, and 356. Theoutput of the photon counters is collected by computer 304, where it canbe stored, analyzed, and viewed on video 360. Alternatively, signaldistribution optics 318 could be a diffraction grating which directsfluorescent signal 318 onto a CCD array.

The stability and reproducibility of the positional localization inscanning will determine, to a large extent, the resolution forseparating closely spaced microparticles. Preferably, the scanningsystems should be capable of resolving closely spaced microparticles,e.g. separated by a particle diameter or less. Thus, for mostapplications, e.g. using CPG microparticles, the scanning system shouldat least have the capability of resolving objects on the order of 10-100μm. Even higher resolution may be desirable in some embodiments, butwith increased resolution, the time required to fully scan a substratewill increase; thus, in some embodiments a compromise may have to bemade between speed and resolution. Increases in scanning time can beachieved by a system which only scans positions where microparticles areknown to be located, e.g from an initial full scan. Preferably,microparticle size and scanning system resolution are selected to permitresolution of fluorescently labeled microparticles randomly disposed ona plane at a density between about ten thousand to one hundred thousandmicroparticles per cm².

In sequencing applications, loaded microparticles can be fixed to thesurface of a substrate in variety of ways. The fixation should be strongenough to allow the microparticles to undergo successive cycles ofreagent exposure and washing without significant loss. When thesubstrate is glass, its surface may be derivatized with an alkylaminolinker using commercially available reagents, e.g. Pierce Chemical,which in turn may be cross-linked to avidin, again using conventionalchemistries, to form an avidinated surface. Biotin moieties can beintroduced to the loaded microparticles in a number of ways. Forexample, a fraction, e.g. 10-15 percent, of the cloning vectors used toattach tags to polynucleotides are engineered to contain a uniquerestriction site (providing sticky ends on digestion) immediatelyadjacent to the polynucleotide insert at an end of the polynucleotideopposite of the tag. The site is excised with the polynucleotide and tagfor loading onto microparticles. After loading, about 10-15 percent ofthe loaded polynucleotides will possess the unique restriction sitedistal from the microparticle surface. After digestion with theassociated restriction endonuclease, an appropriate double strandedadaptor containing a biotin moiety is ligated to the sticky end. Theresulting microparticles are then spread on the avidinated glass surfacewhere they become fixed via the biotin-avidin linkages.

Alternatively and preferably when sequencing by ligation is employed, inthe initial ligation step a mixture of probes is applied to the loadedmicroparticle: a fraction of the probes contain a type IIs restrictionrecognition site, as required by the sequencing method, and a fractionof the probes have no such recognition site, but instead contain abiotin moiety at its non-ligating end. Preferably, the mixture comprisesabout 10-15 percent of the biotinylated probe.

In still another alternative, when DNA-loaded microparticles are appliedto a glass substrate, the DNA may nonspecifically adsorb to the glasssurface upon several hours, e.g. 24 hours, incubation to create a bondsufficiently strong to permit repeated exposures to reagents and washeswithout significant loss of microparticles. Preferably, such a glasssubstrate is a flow cell, which may comprise a channel etched in a glassslide. Preferably, such a channel is closed so that fluids may be pumpedthrough it and has a depth sufficiently close to the diameter of themicroparticles so that a monolayer of microparticles is trapped within adefined observation region.

Parallel Sequencing

The tagging system of the invention can be used with single basesequencing methods to sequence polynucleotides up to several kilobasesin length. The tagging system permits many thousands of fragments of atarget polynucleotide to be sorted onto one or more solid phase supportsand sequenced simultaneously. In accordance with a preferredimplementation of the method, a portion of each sorted fragment issequenced in a stepwise fashion on each of the many thousands of loadedmicroparticles which are fixed to a common substrate--such as amicroscope slide--associated with a scanning system or an image analysissystem, such as described above. The size of the portion of thefragments sequenced depends of several factors, such as the number offragments generated and sorted, the length of the target polynucleotide,the speed and accuracy of the single base method employed, the number ofmicroparticles and/or discrete regions that may be monitoredsimultaneously; and the like. Preferably, from 12-50 bases areidentified at each microparticle or region; and more preferably, 18-30bases are identified at each microparticle or region. With thisinformation, the sequence of the target polynucleotide is determined bycollating the 12-50 base fragments via their overlapping regions, e.g.as described in U.S. Pat. No. 5,002,867. The following referencesprovide additional guidance in determining the portion of the fragmentsthat must be sequenced for successful reconstruction of a targetpolynucleotide of a given length: Lander and Waterman, Genomics, 2:231-239 (1988); Drmanac et al, Genomics, 4: 114-128 (1989); Bains, DNASequencing and Mapping, 4: 143-150 (1993); Bains, Genomics, 11: 294-301(1991); Drmanac et al, J. Biomolecular Structure and Dynamics, 8:1085-1102 (1991); and Pevzner, J. Biomolecular Structure and Dynamics,7: 63-73 (1989). Preferably, the length of the target polynucleotide isbetween 1 kilobase and 50 kilobases. More preferably, the length isbetween 10 kilobases and 40 kilobases. Lander and Waterman (cited above)provide guidance concerning the relationship among the number offragments that are sequenced (i.e. the sample size), the amount ofsequence information obtained from each fragment, and the probabilitythat the target polynucleotide can be reconstructed from the partialsequences without gaps, or "islands." For the present invention, maximalpolynucleotide sizes that can be obtained for given sample sizes andsizes of fragment sequences are shown below:

    ______________________________________                                                Approx. maximal target polynucleotide length                          Size of Sample                                                                          30 bases/fragment                                                                           50 bases/fragment                                     ______________________________________                                         1,000     3 kilobases   4 kilobases                                          10,000    22 kilobases  32 kilobases                                          20,000    40 kilobases  65 kilobases                                          30,000    60 kilobases  85 kilobases                                          100,000   180 kilobases 300 kilobases                                         ______________________________________                                    

Fragments may be generated from a target polynucleotide in a variety ofways, including so-called "directed" approaches where one attempts togenerate sets of fragments covering the target polynucleotide withminimal overlap, and so-called "shotgun" approaches where randomlyoverlapping fragments are generated. Preferably, "shotgun" approaches tofragment generation are employed because of their simplicity andinherent redundancy. For example, randomly overlapping fragments thatcover a target polynucleotide are generated in the followingconventional "shotgun" sequencing protocol, e.g. as disclosed inSambrook et al (cited above). As used herein, "cover" in this contextmeans that every portion of the target polynucleotide sequence isrepresented in each size range, e.g. all fragments between 100 and 200basepairs in length, of the generated fragments. Briefly, starting witha target polynucleotide as an insert in an appropriate cloning vector,e.g. phage, the vector is expanded, purified and digested with theappropriate restriction enzymes to yield about 10-15 μg of purifiedinsert. Typically, the protocol results in about 500-1000 subclones permicrogram of starting DNA. The insert is separated from the vectorfragments by preparative gel electrophoresis, removed from the gel byconventional methods, and resuspended in a standard buffer, such as TE(Tris-EDTA). The restriction enzymes selected to excise the insert fromthe vector preferably leave compatible sticky ends on the insert, sothat the insert can be self-ligated in preparation for generatingrandomly overlapping fragments. As explained in Sambrook et al (citedabove), the circularized DNA yields a better random distribution offragments than linear DNA in the fragmentation methods employed below.After self-ligating the insert, e.g. with T4 ligase using conventionalprotocols, the purified ligated insert is fragmented by a standardprotocol, e.g. sonication or DNase I digestion in the presence of Mn⁺⁺.After fragmentation the ends of the fragments are repair, e.g. asdescribed in Sambrook et al (cited above), and the repaired fragmentsare separated by size using gel electrophoresis. Fragments in the300-500 basepair range are selected and eluted from the gel byconventional means, and ligated into a tag-carrying vector as describedabove to form a library of tag-fragment conjugates.

As described above, a sample containing several thousand tag-fragmentconjugates are taken from the library and expanded, after which thetag-fragment inserts are excised from the vector and prepared forspecific hybridization to the tag complements on microparticles, asdescribed above. Depending of the size of the target polynucleotide,multiple samples may be taken from the tag-fragment library andseparately expanded, loaded onto microparticles and sequenced. Asdiscussed above, the number of doubles selected will depend on thefraction of the tag repertoire represented in a sample. (The probabilityof obtaining triples--three different polynucleotides with the sametag--or above can safely be ignored). As mentioned above, theprobability of doubles in a sample can be estimated from the Poissondistribution p(double)=m² e^(-m) /2, where m is the fraction of the tagrepertoire in the sample. Table VI below lists probabilities ofobtaining doubles in a sample for given tag size, sample size, andrepertoire diversity.

                  TABLE VIII                                                      ______________________________________                                        Number of                    Fraction of                                      words in tag                                                                            Size of tag                                                                             Size of  repertoire                                                                            Probability                              from 8 word set                                                                         repertoire                                                                              sample   sampled of double                                ______________________________________                                        7          2.1 × 10.sup.6                                                                   3000     1.43 × 10.sup.-3                                                                10.sup.-6                                8         1.68 × 10.sup.7                                                                   3 × 10.sup.4                                                                     1.78 × 10.sup.-3                                                                1.6 × 10.sup.-6                                        3000     1.78 × 10.sup.-4                                                                1.6 × 10.sup.-8                    9         1.34 × 108                                                                        3 × 10.sup.5                                                                     2.24 × 10.sup.-3                                                                2.5 × 10.sup.-6                                        3 × 10.sup.4                                                                     2.24 × 10.sup.-4                                                                2.5 × 10.sup.-8                    10        1.07 × 10.sup.-9                                                                  3 × 10.sup.6                                                                      2.8 × 10.sup.-3                                                                3.9 × 10.sup.-6                                        3 × 10.sup.5                                                                      2.8 × 10.sup.-4                                                                3.9 × 10.sup.-8                    ______________________________________                                    

In any case, the loaded microparticles are then dispersed and fixed ontoa glass microscope slide, preferably via an avidin-biotin coupling.Preferably, at least 15-20 nucleotides of each of the random fragmentsare simultaneously sequenced with a single base method. The sequence ofthe target polynucleotide is then reconstructed by collating the partialsequences of the random fragments by way of their overlapping portions,using algorithms similar to those used for assembling contigs, or asdeveloped for sequencing by hybridization, disclosed in the abovereferences.

Kits for Implementing the Method of the Invention

The invention includes kits for carrying out the various embodiments ofthe invention. Preferably, kits of the invention include a repertoire oftag complements attached to a solid phase support. Additionally, kits ofthe invention may include the corresponding repertoire of tags, e.g. asprimers for amplifying the polynucleotides to be sorted or as elementsof cloning vectors which can also be used to amplify the polynucleotidesto be sorted. Preferably, the repertoire of tag complements are attachedto microparticles. Kits may also contain appropriate buffers forenzymatic processing, detection chemistries, e.g. fluorescent orchemiluminescent tags, and the like, instructions for use, processingenzymes, such as ligases, polymerases, transferases, and so on. In animportant embodiment for sequencing, kits may also include substrates,such as a avidinated microscope slides, for fixing loaded microparticlesfor processing.

Identification of Novel Polynucleotides in cDNA Libraries

Novel polynucleotides in a cDNA library can be identified byconstructing a library of cDNA molecules attached to microparticles, asdescribed above. A large fraction of the library, or even the entirelibrary, can then be partially sequenced in parallel. After isolation ofmRNA, and perhaps normalization of the population as taught by Soares etal, Proc. Natl. Acad. Sci., 91: 9228-9232 (1994), or like references,the following primer (SEQ ID NO:9) may by hybridized to the polyA tailsfor first strand synthesis with a reverse transcriptase usingconventional protocols:

    ______________________________________                                        5'-mRNA-                                                                             [A].sub.n -3'                                                                 [T].sub.19 -[primer site]-GG[W,W,W,C].sub.9 ACCAGCTGATC-5'             ______________________________________                                    

where [W,W,W,C]₉ represents a tag as described above, "ACCAGCTGATC" isan optional sequence (SEQ ID NO:10) forming a restriction site in doublestranded form, and "primer site" is a sequence common to all members ofthe library that is later used as a primer binding site for amplifyingpolynucleotides of interest by PCR.

After reverse transcription and second strand synthesis by conventionaltechniques, the double stranded fragments are inserted into a cloningvector as described above and amplified. The amplified library is thensampled and the sample amplified. The cloning vectors from the amplifiedsample are isolated, and the tagged cDNA fragments excised and purified.After rendering the tag single stranded with a polymerase as describedabove, the fragments are methylated and sorted onto microparticles inaccordance with the invention. Preferably, as described above, thecloning vector is constructed so that the tagged cDNAs can be excisedwith an endonuclease, such as Fok I, that will allow immediatesequencing by the preferred single base method after sorting andligation to microparticles.

Stepwise sequencing is then carried out simultaneously on the wholelibrary, or one or more large fractions of the library, in accordancewith the invention until a sufficient number of nucleotides areidentified on each cDNA for unique representation in the genome of theorganism from which the library is derived. For example, if the libraryis derived from mammalian mRNA then a randomly selected sequence 14-15nucleotides long is expected to have unique representation among the 2-3thousand megabases of the typical mammalian genome. Of courseidentification of far fewer nucleotides would be sufficient for uniquerepresentation in a library derived from bacteria, or other lowerorganisms. Preferably, at least 20-30 nucleotides are identified toensure unique representation and to permit construction of a suitableprimer as described below. The tabulated sequences may then be comparedto known sequences to identify unique cDNAs.

Unique cDNAs are then isolated by conventional techniques, e.g.constructing a probe from the PCR amplicon produced with primersdirected to the prime site and the portion of the cDNA whose sequencewas determined. The probe may then be used to identify the cDNA in alibrary using a conventional screening protocol.

The above method for identifying new cDNAs may also be used tofingerprint mRNA populations, either in isolated measurements or in thecontext of a dynamically changing population. Partial sequenceinformation is obtained simultaneously from a large sample, e.g. ten toa hundred thousand, or more, of cDNAs attached to separatemicroparticles as described in the above method. The frequencydistribution of partial sequences can identify mRNA populations fromdifferent cell or tissue types, as well as from diseased tissues, suchas cancers. Such mRNA fingerprints are useful in monitoring anddiagnosing disease states, e.g. International applicationPCT/US95/21944, which describes the use of express sequence tags (ESTs)for the same purpose.

Cycle Sequencing on Microparticles Loaded with Sorted Polynucleotides

Parallel sequencing may also be accomplished in accordance with theinvention with conventional sequencing techniques that require thegeneration and separation of labeled DNA fragments. In particular,isolated microparticles loaded with a uniform population of templatesmay be used to generate labeled extension products by cycle sequencing.Cycle sequencing is a well-know variant of the basic Sanger approach toDNA sequencing describe fully in the following references: Craxton,Methods, Vol. 2 (February, 1991); Wozny, European patent publication 0409 078 A2 (Jan. 23, 1991); Fuller, International applicationPCT/US92/07303; and Fuller, International application PCT/US94/03264.Briefly, in a standard sequencing reaction mixture, a thermal stablepolymerase is employed so that repeated extension reactions may becarried out on the same template. This permits small amounts of templateto generate sufficient amounts of extension product for detection afterseparation by electrophoresis. Typically, cycle sequencing comprises thesteps of (a) providing a sequencing reaction mixture with a template, aprimer, nucleoside triphosphates, chain-terminating nucleosidetriphosphates, and a thermal stable DNA polymerase; (b) denaturing thetemplate, (c) annealing the primer to the denatured template, (d)extending the primer to form extension products, and (e) repeating steps(b)-(d) until sufficient quantities of extension products areaccumulated so that they may be detected upon separation. The number oftimes the cycle is repeated depends on many factors, including theamount and quality of starting template, the detection system employed,the separation system employed, and the like. As conventionallypracticed, the extension cycle is typically repeated from 10 to 100times; the template amount ranges from as little as a few tens offemtomole to several tens of picomole; the denaturation step is carriedout by heating the reaction mixture to a temperature in the range of92-95° C.; the annealing step takes place at a temperature in the rangeof 35-75° C.; and the extension step takes place at a temperature in therange of 65-85° C. with a thermal stable DNA polymerase, such as Taq orVent (available from Perkin-Elmer Corp., Norwalk, Conn., and New EnglandBiolabs, respectively).

Tag complements may be prepared on magnetic microparticles as describedby Albretsen et al, Anal. Biochem., 189: 40-50 (1990), which allowsloadings of several femtomoles of tag complements onto 4.5 μm diametermagnetic beads. Tag complements may be attached to the microparticleseither by their 5' or 3' ends. If attached by 5' ends, then thetemplates may be sorted via specific hybridization of tags at their 3'ends. In this embodiment, the template has a primer complement at its 5'end, as shown below:

    3'-[oligonucleotide tag]-[template]-[primer complement]-5'

The tag complement is then extended the length of the template so that acomplement of the template is obtained which is covalently attached tothe microparticle. The template is removed by heating and themicroparticles are washed. After microparticles are separated, e.g. byflow sorting, repeated cycles of annealing primers, extension, anddenaturation are carried out.

If tag complements are attached to the microparticles by their 3' ends,which allows for convenient synthesis directly on the microparticles,the order of the oligonucleotide tag and primer complement are reversed,as shown below:

    5'-[oligonucleotide tag]-[template]-[primer complement]-3'

Also, the 5' end of the tag complement is phosphorylated, e.g. usingcommercially available reagents. After specific hybridization via theoligonucleotide tag, a primer is annealed to the primer complement atthe 3' end of the template and extended with a DNA polymerase lacking3'→45' exonuclease activity. The nick left by this extension reaction isthen ligated and the original template removed by heating. Afterseparating microparticles, the cycle sequencing can be carried out asabove.

Separation of loaded microparticles may be carried out by flow sorting,wherein suspended microparticles are entrained to pass single filethrough a nozzle and in a liquid jet which is broken up into a regularseries of charged droplets which are directed to predetermined targetvessels, wells, or other reaction locations on a substrate.Microparticles are conveniently detected in the jet by light scatter andthe magnitude of the scatter is used to determine whether a dropletcontains no, one, or multiple microparticles. A particularly usefulapparatus for such flow sorting and delivery of sequencing reagent isdisclosed in Brennan, International application PCT/US94/05896. Once theindividual loaded microparticles are distributed to a plurality ofreaction sites or wells with the appropriate sequencing reagents, thecollection of reactions can be thermally cycled together to generateextension products. After cycling is completed, the extension productsare separated by electrophoresis. Preferably, electrophoretic separationis carried out by capillary electrophoresis in a gel-free separationmedium, which allows convenient loading and rapid separation of theextension fragments. Also, apparatus is available which permitsdetection by four-color fluorescence of a large number of samplessubstantially at the same time, e.g. the type disclosed by Mathies andHuang, Nature, 359:167-169 (1992); Huang et al, Anal. Chem., 64:2149-2154 (1992); Huang et al, Anal. Chem., 64: 967-972 (1992); or thelike. Preferably, several thousand cycle sequencing reactions arecarried at the same time. More preferably, mixtures of templates aresorted onto a population of microparticles having a repertoire ofoligonucleotide tags of between 1000 and 10,000 different types.

Sorting Multi-locus Probes for Genotypic Analysis

Many disease conditions and/or disease susceptibilities are associatedwith complex genetic traits and/or patterns of mutation, e.g. HLA type,mutation pattern of the p53 gene in many cancers, the cystic fibrosisgene, Lesch-Nyhan syndrome, Duchenne muscular dystrophy, and the like,Lander et al, Science, 265: 2037-2048 (1994); Collins, Science,256:774-779 (1992); Tsui et al, International patent applicationPCT/CA90/00267; Hedrum et al, Biotechniques, 17: 118-129 (1994);Santamaria et al, International patent application PCT/US92/01675;Chamberlain et al, Nucleic Acids Research, 16: 11141-11156 (1988); andthe like. One approach to constructing convenient assays for suchcomplex genetic traits has been to use so-called multiplex PCR ormultiplex ligation assays, such as described in Chamberlain et al (citedabove) or in Grossman et al, International patent applicationPCT/US93/03229. Usually, such techniques call for the simultaneousamplification of multiple genetic sequences in the same reaction mixturefollowed by specific detection of sequences of interest. Oligonucleotidetags of the invention can provide a simple and convenient means foridentifying genetic sequences that are amplified in such assays. In itssimplest form, this embodiment of the invention may be implement byattaching oligonucleotide tags to PCR primers used in multiplex PCR. Oneprimer of a pair carries an oligonucleotide tag and the other primer ofthe pair carries a capture moiety, such as described above, that permitsisolation and then release of successfully amplified sequences. Afterrelease, the sequences are applied to solid phase support having a setof tag complements attached at predefined spatial addresses. The patternof specific hybridization of the tags is then detected to identify thegenotype of a sample.

In a preferred embodiment, PCR is employed to amplify a geneticsequences of interest that contains multiple target sites, i.e. multiplesites where mutations or disease-related sequences occur. Preferably,only two or very few pairs of primers are used to amplify the targetsequence to avoid the difficulties involved with multiplex PCR, such asbalancing target lengths, primer annealing temperatures, and the like.After amplification, specific genotypes are detected in a manneranalogous to that described in Grossman et al (cited above) and Grossmanet al, U.S. Pat. No. 5,514,543, which references provide guidance in theselection of PCR and ligation reaction conditions, ligation probe sizes,and the like. In those references, a target sequence is similarlyamplified, after which a collection of ligation probes are applied inthe presence of a DNA ligase. The ligation probes consist of twoseparate sequences both complementary to a target potentially present ina sample being analyzed: one is attached to a electrophoretic mobilitymodifier and the other is attached to a fluorescent label. If the twoprobes form perfect duplexes with the target sequence in the sample theyare ligated so that the mobility modifying moiety is now attached to afluorescent label through the ligated sequences complementary to thetarget. The components of the mixture are then separatedelectrophoretically so that the pattern of fluorescent bands on a gel isindicative of the genotype of the target present in the sample. As shownin FIG. 3, oligonucleotide tags of the invention may be used in place ofthe electrophoretic mobility modifiers and spatial separation can beachieved by sorting ligated sequences to particular locations on a solidphase support. Returning to FIG. 3, target sequence (200) is amplified,preferably by PCR, after which a collection of ligation probes (206-216)is applied (204) to a denatured amplicon. In this embodiment, ligationprobes comprise an oligonucleotide tag (206), a first sequence (208)complementary to a target sequence, a second sequence (210)complementary to the target sequence and contiguous with the firstsequence (such that if both are perfectly complementary to the targetsequence they are capable of being ligated), a tail (212) carrying asignal generating means (214). Signal generating means (214) ispreferably a fluorescent label. Preferably, the first and secondsequences of the ligation probes are ligated by a DNA ligase; thus, the5' end of the abutting sequences (216) must be phosphorylated, e.g. viaa phosphorylating reagent described in Urdea et al, U.S. Pat. No.5,332,845. After application of the ligation probes and a ligase, probesforming perfectly matched duplexes with the target sequence arecovalently joined (218 & 220). The probe-target duplexes are thendenatured and applied (222) to a solid phase support which has tagcomplement attached at well defined spatial locations for every tag t₁through t_(k). After washing off nonspecifically bound sequences, thespatial locations corresponding to the tag complements of theoligonucleotide tags, t_(i) and t_(j), which were ligated to fluorescentlabels are illuminated, as shown in FIG. 3 by 226 and 228. The patternof illuminated fluorophors on the solid phase support indicates thegenotype of the target sequence in the sample. Preferably, in thisembodiment of the invention there is a one-to-one correspondence betweena tag and a spatial address on the solid phase support. In furtherpreference, this embodiment is employed to simultaneously identify atleast twenty gene targets; and more preferably, it is employed tosimultaneously detect at least 50 gene targets.

Generally, this embodiment of the invention may be with the followingsteps for detecting the presence or absence of a plurality of selectedtarget sequences in a target polynucleotide: (1) adding to the targetpolynucleotide a plurality of ligation probes, each ligation probeincluding a first oligonucleotide and a second oligonucleotide which arecomplementary in sequence to adjacent portions of a selected one of thetarget sequences in the target polynucleotide, the first oligonucleotidehaving an oligonucleotide tag attached, each oligonucleotide tag beingselected from the same minimally cross-hybridizing set and each ligationprobe having a different oligonucleotide tag; (2) hybridizing the probeswith the target polynucleotide; (3) treating the hybridized first andsecond oligonucleotides under conditions effective to ligate the firstand second oligonucleotides whenever the first and second oligos formperfectly match duplexes with adjacent target sequences; (4) separatingligated first and second oligos from unligated first and secondoligonucleotides; (5) sorting the ligated first and secondoligonucleotides by specifically hybridizing the oligonucleotide tagswith their respective complements, the respective complements beingattached as uniform populations of substantially identicaloligonucleotides in spatially discrete regions on the one or more solidphase supports; and (6) detecting the presence or absence of theselected target sequences by the presence or absence of ligated firstand second oligonucleotides on the one or more solid phase supports.

EXAMPLE 1 Sorting Multiple Target Polynucleotides Derived from pUC19

A mixture of three target polynucleotide-tag conjugates are obtained asfollows: First, the following six oligonucleotides are synthesized andcombined pairwise to form tag 1, tag 2, and tag 3 (SEQ ID NO:11, SEQ IDNO:12, and SEQ ID: 13):5'-pTCGACC(w1)(w2)(w3)(w4)(w5)(w6)(w7)(w8)(w1)ATag 1GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5'5'-pTCGACC(w6)(w7)(w8)(w1)(w2)(w6)(w4)(w2)(w1)ATag 2GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5'5'-pTCGACC(w3)(w2)(w1)(w1)(w5)(w8)(w8)(w4)(w4)ATag 3 GG(**)(**)(**)(**)(**)(**)(**)(**)(**)ttcgaP-5'

where "p" indicates a monophosphate, the w_(i) 's represent the subunitsdefine in Table II, and the terms "(**)" represent their respectivecomplements. A pUC19 is digested with Sal I and Hind III, the largefragment is purified, and separately ligated with tags 1, 2, and 3, toform pUC19-1, pUC19-2, and pUC19-3, respectively. The three recombinantsare separately amplified and isolated, after which pUC19-1 is digestedwith Hind III and Aat I, pUC19-2 is digested with Hind III and Ssp I,and pUC19-3 is digested with Hind III and Xmn I. The small fragments areisolated using conventional protocols to give three double strandedfragments about 250, 375, and 575 basepairs in length, respectively, andeach having a recessed 3' strand adjacent to the tag and a blunt or 3'protruding strand at the opposite end. Approximately 12 nmoles of eachfragment are mixed with 5 units T4 DNA polymerase in the manufacturer'srecommended reaction buffer containing 33 M deoxycytosine triphosphate.The reaction mixture is allowed to incubate at 37° C. for 30 minutes,after which the reaction is stopped by placing on ice. The fragments arethen purified by conventional means.

CPG microparticles (37-74 mm particle size, 500 angstrom pore size,Pierce Chemical) are derivatized with the linker disclosed by Maskos andSouthern, Nucleic Acids Research, 20: 1679-1684 (1992). After separatinginto three aliquots, the complements of tags 1, 2, and 3 are synthesizedon the microparticles using a conventional automated DNA synthesizer,e.g. a model 392 DNA synthesizer (Applied Biosystems, Foster City,Calif.). Approximately 1 mg of each of the differently derivatizedmicroparticles are placed in separate vessels.

The T4 DNA polymerase-treated fragments excised from pUC19-1, -2, and -3are resuspended in 50 L of the manufacturer's recommended buffer for TaqDNA ligase (New England Biolabs). The mixture is then equally dividedamong the three vessels containing the 1 mg each of derivatized CPGmicroparticles. 5 units of Taq DNA ligase is added to each vessel, afterwhich they are incubated at 55° C. for 15 minutes. The reaction isstopped by placing on ice and the microparticles are washed severaltimes by repeated centrifugation and resuspension in TE. Finally, themicroparticles are resuspended in Nde I reaction buffer (New EnglandBiolabs) where the attached polynucleotides are digested. Afterseparation from the microparticles the polynucleotide fragments releasedby Nde I digestion are fluorescently labeled by incubating withSequenase DNA polymerase and fluorescein labeled thymidine triphosphate(Applied Biosystems, Foster City, Calif.). The fragments are thenseparately analyzed on a nondenaturing polyacrylamide gel using anApplied Biosystems model 373 DNA sequencer.

EXAMPLE 2 Parallel Sequencing of SV40 Fragments

A repertoire of 36-mer tags consisting of nine 4-nucleotide subunitsselected from Table II is prepared by separately synthesizing tags andtag complements by a split and mix approach, as described above. Therepertoire is synthesized so as to permit ligation into a Sma I/Hind IIIdigested M13mp19. Thus, as in Example 1, one set of oligonucleotidesbegins with the addition of A followed by nine rounds of split and mixsynthesis wherein the oligonucleotide is extended subunit-wise by3'-phosphoramidite derivatived 4-mers corresponding to the subunits ofTable II. The synthesis is then completed with thenucleotide-by-nucleotide addition of one half of the Sma I recognitionsite (GGG), two C's, and a 5'-monophosphate, e.g. via the Phosphate-ONreagent available from Clontech Laboratories (Palo Alto, Calif.). Theother set of oligonucleotides begins with the addition of three C's(portion of the Sma I recognition site) and two G's, followed by ninerounds of split and mix synthesis wherein the oligonucleotide isextended by 3'-phosphoramidite derivatized 4-mers corresponding to thecomplements of the subunits of Table II. Synthesis is completed by thenucleotide-by-nucleotide addition of the Hind III recognition site and a5'-monophosphate. After separation from the synthesis supports theoligonucleotides are mixed under conditions that permit formation of thefollowing duplexes: (SEQ IDNO:14)5'-pGGGCC(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)ACCCGG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5'

The mixture of duplexes is then ligated into a Sma I/Hind III-digestedM13mp19. A repertoire of tag complements are synthesized on CPGmicroparticles as described above.

Next the following adaptor (SEQ ID NO:15) is prepared which contains aFok I site and portions of Eco RI and Sma Isites:5'-pAATTCGGATGATGCATGCATCGACCC GCCTACTACGTACGTAGCTGGGp-5'Eco RIFok I Sma I

The adaptor is ligated into the Eco RI/Sma I digested M13 describedabove.

Separately, SV40 DNA is fragmented by sonication following the protocolset forth in Sambrook et al (cited above). The resulting fragments arerepaired using standard protocols and separated by size. Fragments inthe range of 300-500 basepairs are selected and ligated into the Sma Idigested M13 described above to form a library of fragment-tagconjugates, which is then amplified. A sample containing severalthousand different fragment-tag conjugates is taken from the library,further amplified, and the fragment-tag inserts are excised by digestingwith Eco RI and Hind III. The excised fragment-tag conjugates aretreated with T4 DNA polymerase in the presence of deoxycytidinetriphosphate, as described in Example 1, to expose the oligonucleotidetags for specific hybridization to the CPG microparticles.

After hybridization and ligation, as described in Example 1, the loadedmicroparticles are treated with Fok I to produce a 4-nucleotideprotruding strand of a predetermined sequence. A 10:1 mixture (probe1:probe 2) of the following probes (SEQ ID NO:16 and SEQ ID NO:17) areligated to the polynucleotides on microparticles.Probe 1\\\FAM-ATCGGATGAC \\\\\\\TAGCCTACTGAGCTProbe 2 biotin-ATCCAATGAC\\\\\\\TAGGTTACTGAGCT

FAM represents a fluorescein dye attached to the 5'-hydroxyl of the topstrand of Probe 1 through an aminophosphate linker available fromApplied Biosystems (Aminolinker). The biotin may also be attachedthrough an Aminolinker moiety and optionally may be further extended viapolyethylene oxide linkers, e.g. Jaschke et al (cited above).

The loaded microparticles are then deposited on the surface of anavidinated glass slide to which and from which reagents and washsolutions can be delivered and removed. The avidinated slide with theattached microparticles is examined with a scanning fluorescentmicroscope (e.g. Zeiss Axioskop equipped with a Newport Model PM500-Cmotion controller, a Spectra-Physics Model 2020 argon ion laserproducing a 488 nm excitation beam, and a 520 nm long-pass emissionfilter, or like apparatus). The excitation beam and fluorescentemissions are delivered and collected, respectively, through the sameobjective lens. The excitation beam and collected fluorescence areseparated by a dichroic mirror which directs the collected fluorescencethrough a series of bandpass filters and to photon-counting devicescorresponding to the fluorophors being monitored, e.g. comprisingHamamatsu model 9403-02 photomultipliers, a Stanford Research Systemsmodel SR445 amplifier and model SR430 multichannel scaler, and digitalcomputer, e.g. a 486-based computer. The computer generates a twodimensional map of the slide which registers the positions of themicroparticles.

After cleavage with Fok I to remove the initial probe, thepolynucleotides on the attached microparticles undergo 20 cycles ofprobe ligation, washing, detection, cleavage, and washing, in accordancewith the preferred single base sequencing methodology described below.Within each detection step, the scanning system records the fluorescentemission corresponding the base identified at each microparticle.Reactions and washes below are generally carried out with manufacturer's(New England Biolabs') recommended buffers for the enzymes employed,unless otherwise indicated. Standard buffers are also described inSambrook et al (cited above).

The following four sets of mixed probes (SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, and SEQ ID NO:21) are provided for addition to the targetpolynucleotides: TAMRA-ATCGGATGACATCAAC TAGCCTACTGTAGTTGANNNFAM-ATCGGATGACATCAAC TAGCCTACTGTAGTTGCNNN ROX-ATCGGATGACATCAACTAGCCTACTGTAGTTGGNNN JOE-ATCGGATGACATCAAC TAGCCTACTGTAGTTGTNNN

where TAMRA, FAM, ROX, and JOE are spectrally resolvable fluorescentlabels attached by way of Aminolinker II (all being available fromApplied Biosystems, Inc., Foster City, Calif.); the bold facednucleotides are the recognition site for Fok I endonuclease, and "N"represents any one of the four nucleotides, A, C, G, T. TAMRA(tetramethylrhodamine), FAM (fluorescein), ROX (rhodamine X), and JOE(2',7'-dimethoxy-4',5'-dichlorofluorescein) and their attachment tooligonucleotides is also described in Fung et al, U.S. Pat. No.4,855,225.

The above probes are incubated in approximately 5 molar excess of thetarget polynucleotide ends as follows: the probes are incubated for 60minutes at 16° C. with 200 units of T4 DNA ligase and the anchoredtarget polynucleotide in T4 DNA ligase buffer; after washing, the targetpolynucleotide is then incubated with 100 units T4 polynucleotide kinasein the manufacturers recommended buffer for 30 minutes at 37° C.,washed, and again incubated for 30 minutes at 16° C. with 200 units ofT4 DNA ligase and the anchored target polynucleotide in T4 DNA ligasebuffer. Washing is accomplished by successively flowing volumes of washbuffer over the slide, e.g. TE, disclosed in Sambrook et al (citedabove). After the cycle of ligation-phosphorylation-ligation and a finalwashing, the attached microparticles are scanned for the presence offluorescent label, the positions and characteristics of which arerecorded by the scanning system. The labeled target polynucleotide, i.e.the ligated complex, is then incubated with 10 units of Fok I in themanufacturer's recommended buffer for 30 minutes at 37° C., followed bywashing in TE. As a result the target polynucleotide is shortened by onenucleotide on each strand and is ready for the next cycle of ligationand cleavage. The process is continued until twenty nucleotides areidentified.

EXAMPLE 3 Construction of a Tag Library

An exemplary tag library is constructed as follows to form thechemically synthesized 9-word tags (SEQ ID NO:22) of nucleotides A, G,and T defined by the formula:

    3'-TGGC-[.sup.4(A,G,T).sub.9 ]-CCCCp

where "[⁴((A,G,T)₉ ]" indicates a tag mixture where each tag consists ofnine 4-mer words of A, G, and T; and "p" indicate a 5' phosphate. Thismixture is ligated to the following right and left primer bindingregions: (SEQ ID NO:23 and SEQ ID NO:24)5'-AGTGGCTGGGCATCGGACCG5'-GGGGCCCAGTCAGCGTCGAT TCACCGACCCGTAGCCp GGGTCAGTCGCAGCTALeft Right

The right and left primer binding regions are ligated to the above tagmixture (SEQ ID NO:22), after which the single stranded portion of theligated structure is filled with DNA polymerase then mixed with theright and left primers indicated below (SEQ ID NO:25 and SEQ ID NO:26)and amplified to give a tag library. Left Primer5'-AGTGGCTGGGCATCGGACCG5'- AGTGGCTGGGCATCGGACCG- [⁴( (A,G,T)₉]-GGGGCCCAGTCAGCGTCGAT TCACCGACCCGTAGCCTGGC- [⁴( (A,G,T)₉]-CCCCGGGTCAGTCGCAGCTA CCCCGGGTCAGTCGCAGCTA-5' Right Primer

The underlined portion of the left primer binding region indicates a RsrII recognition site. The left-most underlined region of the right primerbinding region indicates recognition sites for Bsp 120I, Apa I, and EcoO 109I, and a cleavage site for Hga I. The right-most underlined regionof the right primer binding region indicates the recognition site forHga I. Optionally, the right or left primers may be synthesized with abiotin attached (using conventional reagents, e.g. available fromClontech Laboratories, Palo Alto, Calif.) to facilitate purificationafter amplification and/or cleavage.

EXAMPLE 4 Construction of a Plasmid Library of Tag-PolynucleotideConjugates for cDNA "Signature" Sequencing

cDNA is produced from an mRNA sample by conventional protocols usingpGGCCCT₁₅ (A or G or C) as a primer for first strand synthesis anchoredat the boundary of the poly A region of the mRNAs and N₈ (A or T)GATC asthe primer for second strand synthesis. That is, both are degenerateprimers such that the second strand primer is present in two forms andthe first strand primer is present in three forms. The GATC sequence inthe second strand primer corresponds to the recognition site of Mbo I;other four base recognition sites could be used as well, such as thosefor Bam H1, Sph I, Eco RI, or the like. The presence of the A and Tadjacent to the restriction site of the second stand primer ensures thata stripping and exchange reaction can be used in the next step togenerate a five-base 5' overhang of "GGCCC". The first strand primer isannealed to the mRNA sample and extended with reverse transcriptase,after which the RNA strand is degraded by the RNase H activity of thereverse transcriptase leaving a single stranded cDNA. The second strandprimer is annealed and extended with a DNA polymerase using conventionalprotocols. After second strand synthesis, the resulting cDNAs aremethylated with CpG methylase (New England Biolabs, Beverly, Mass.)using manufacturer's protocols. The 3' strands of the cDNAs are then cutback with the above-mentioned stripping and exchange reaction using T4DNA polymerase in the presence of dATP and dTTP, after which the cDNAsare ligated to the tag library of Example 3 previously cleaved with HgaI to give the following construct: ##STR1## Separately, the followingcloning vector (SEQ ID NO:27) is constructed, e.g. starting from acommercially available plasmid, such as a Bluescript phagemid(Stratagene, La Jolla, Calif.). ##STR2## The plasmid is cleaved with PpuMI and Pme I (to give a Rsr II-compatible end and a flush end so thatthe insert is oriented) and then methylated with DAM methylase. Thetag-containing construct is cleaved with Rsr II and then ligated to theopen plasmid, after which the conjugate is cleaved with Mbo I and Bam HIto permit ligation and closing of the plasmid. The plasmid is thenamplified and isolated and used in accordance with the invention.

    __________________________________________________________________________    APPENDIX Ia                                                                   Exemplary computer program for generating                                     minimally cross hybridizing sets                                              (single stranded tag/single stranded tag complement)                          __________________________________________________________________________    Program minxh                                                                 c                                                                             c                                                                             integer*2 sub1(6) ,mset1(1000,6),mset2(1000,6)                                dimension nbase(6)                                                            c                                                                             write(*,*) 'ENTER SUBUNIT LENGTH'                                             read(*,100)nsub                                                               100   format(i1)                                                              open (1,file='sub4.dat',form='formatted',status='new')                        c                                                                             c                                                                             nset=0                                                                        do 7000 ml=1,3                                                                        do 7000 m2=1,3                                                                  do 7000 m3=1,3                                                                  do 7000 m4=1,3                                                                  sub1(1) =m1                                                                   sub1(2) =m2                                                                   sub1(3) =m3                                                                   sub1(4) =m4                                                     c                                                                             c                                                                             ndiff=3                                                                       c                                                                             c                                                                             c           Generate set of subunits differing from                           c           sub1 by at least ndiff nucleotides.                               c           Save in mset1.                                                    c                                                                             c                                                                             jj=1                                                                          do 900 j=1,nsub                                                               900     mset1(1,j)=sub1(j)                                                    c                                                                             c                                                                             do 1000 k1=1,3                                                                        do 1000 k2=1,3                                                                  do 1000 k3=1,3                                                                  do 1000 k4=1,3                                                    c                                                                             c                                                                                         nbase (1)=k1                                                                  nbase (2)=k2                                                                  nbase (3)=k3                                                                  nbase (4)=k4                                                      c                                                                                         n=0                                                                           do 1200 j=1,nsub                                                                if(sub1(j).eq.1 .and. nbase(j) .ne.1 .or.                       1               sub1(j).eq.2 .and. nbase(j) .ne.2 .or.                        3               sub1(j).eq.3 .and. nbase(j) .ne.3) then                                       n=n+1                                                                         endif                                                         1200            continue                                                      c                                                                             c                                                                             if(n.ge.ndiff) then                                                           c                                                                             c                                                                             c                 If number of mismatches                                     c                 is greater than or equal                                    c                 to ndiff then record                                        c                 subunit in matrix mset                                      c                                                                             c                                                                                       jj=jj+1                                                                         do 1100 i=1,nsub                                                  1100          mset1(jj,i)=nbase(i)                                                      endif                                                               c                                                                             c                                                                             1000  continue                                                                c                                                                             c                                                                                     do 1325 j2=1,nsub                                                             mset2(1,j2)=mset1(1,j2)                                               1325    mset2(2,j2)=mset1(2,j2)                                               c                                                                             c                                                                             c                 Compare subunit 2 from                                      c                 mset1 with each successive                                  c                 subunit in mset1, i.e. 3,                                   c                 4,5, . . . etc. Save those                                  c                 with mismatches .ge. ndiff                                  c                 in matrix mset2 starting at                                 c                 position 2.                                                 c                   Next transfer contents                                    c                 of mset2 into mset1 and                                     c                 start                                                       c                 comparisons again this time                                 c                 starting with subunit 3.                                    c                 Continue until all subunits                                 c                 undergo the comparisons.                                    c                                                                             c                                                                             npass=0                                                                       c                                                                             c                                                                             1700  continue                                                                kk=npass+2                                                                    npass=npass+1                                                                 c                                                                             do 1500 m=npass+2,jj                                                                  n=0                                                                           do 1600 j=1,nsub                                                                if(mset1(npass+1,j) .eq.1.and.mset1(m,j) .ne.1.or.                  2           mset1(npass+1,j) .eq.2.and.mset1(m,j) .ne.2.or.                   2           mset1(npass+1,j) .eq.3.and.mset1(m,j) .ne.3) then                             n=n+1                                                                         endif                                                             1600        continue                                                                    if(n.ge.ndiff) then                                                               kk=kk+1                                                                       do 1625 i=1,nsub                                                1625            mset2 (kk,i)=mset1(m,i)                                                 endif                                                               1500      continue                                                            c                                                                             c                 kk is the number of subunits                                c                 stored in mset2                                             c                                                                             c                 Transfer contents of mset2                                  c                 into mset1 for next pass.                                   c                                                                             c                                                                                     do 2000 k=1,kk                                                                  do 2000 m=1,nsub                                                    2000        mset1(k,m)=mset2(k,m)                                             if(kk.1t.jj) then                                                                     jj=kk                                                                         goto 1700                                                                     endif                                                                 c                                                                             c                                                                                     nset=nset+1                                                           write (1,7009)                                                                7009    format(/)                                                             do 7008 k=1,kk                                                                7008      write(1,7010) (mset1(k,m),m=1,nsub)                                 7010  format(4i1)                                                             write (*,*)                                                                   write(*,120) kk,nset                                                          120   format(1x,'Subunits in set=',i5,2x,'Set No=',i5)                        7000    continue                                                                      close (1)                                                             c                                                                             c                                                                             end                                                                           c           **********************************                                c           **********************************                                __________________________________________________________________________

    __________________________________________________________________________    APPENDIX Ib                                                                   Exemplary computer program for generating                                     minimally cross hybridizing sets                                              (single stranded tag/single stranded tag complement)                          __________________________________________________________________________    Program tagN                                                                  c                                                                             c     Program tagN generates minimally cross-hybridizing                      c       sets of subunits given i) N--subunit length, and ii)                  c       an initial subunit sequence. tagN assumes that only                   c       3 of the four natural nucleotides are used in the tags.               c                                                                             c                                                                             character*i sub1(20)                                                          integer*2 mset(10000,20), nbase(20)                                           c                                                                             c                                                                             write(*,*) 'ENTER SUBUNIT LENGTH'                                             read(*,100)nsub                                                               100 format(i2)                                                                c                                                                             c                                                                             write (*,*) 'ENTER SUBUNIT SEQUENCE'                                          read(*,110) (sub1(k) ,k=1,nsub)                                               110 format(20a1)                                                              c                                                                             c                                                                             ndiff=10                                                                      c                                                                             c                                                                                       Let a=1 c=2 g=3 & t=4                                               c                                                                             c                                                                             do 800 kk=1,nsub                                                              if(sub1(kk) .eq. 'a') then                                                            mset(1, kk)=1                                                                 endif                                                                           if(sub1(kk) .eq. 'c') then                                                      mset (1,kk)=2                                                                 endif                                                                           if(sub1(kk) .eq. 'g') then                                                      mset (1, kk)=3                                                                endif                                                                           if(sub1(kk) .eq. 't') then                                                      mset (1,kk)=4                                                                 endif                                                     800 continue                                                                  c                                                                             c                                                                             c         Generate set of subunits differing from                             c         sub1 by at least ndiff nucleotides.                                 c                                                                             c                                                                             jj=1                                                                          c                                                                             c                                                                             do 1000 k1=1,3                                                                        do 1000 k2=1,3                                                                  do 1000 k3=1,3                                                                  do 1000 k4=1,3                                                                  do 1000 k5=1,3                                                                  do 1000 k6=1,3                                                                  do 1000 k7=1,3                                                                  do 1000 k8=1,3                                                                  do 1000 k9=1,3                                                                  do 1000 k10=1,3                                       do 1000 k11=1,3                                                                       do 1000 k12=1,3                                                                 do 1000 k13=1,3                                                                 do 1000 k14=1,3                                                                 do 1000 k15=1,3                                                                 do 1000 k16=1,3                                                                 do 1000 k17=1,3                                                                 do 1000 k18=1,3                                                                 do 1000 k19=1,3                                                                 do 1000 k20=1,3                                       c                                                                             c                                                                                         nbase(1)=k1                                                                   nbase(2)=k2                                                                   nbase(3)=k3                                                                   nbase(4)=k4                                                                   nbase(5)=k5                                                                   nbase(6)=k6                                                                   nbase(7)=k7                                                                   nbase(8)=k8                                                                   nbase(9)=k9                                                                   nbase(10)=k10                                                                 nbase(11)=k11                                                                 nbase(12)=k12                                                                 nbase(13)=k13                                                                 nbase(14)=k14                                                                 nbase(15)=k15                                                                 nbase(16)=k16                                                                 nbase(17)=k17                                                                 nbase(18)=k18                                                                 nbase(19)=k19                                                                 nbase(20)=k20                                                     c                                                                             c                                                                                     do 1250 nn=1,jj                                                       c                                                                                       n=0                                                                           do 1200 j=1,nsub                                                                  if(mset (nn,j) .eq.1 .and. nbase(j) .ne.1 .or.                  1               mset(nn,j) .eq.2 .and. nbase(j) .ne.2 .or.                    2               mset(nn,j) .eq.3 .and. nbase(j) .ne.3 .or.                    3               mset(nn,j) .eq.4 .and. nbase(j) .ne.4) then                                   n=n+1                                                                         endif                                                         1200            continue                                                      c                                                                             c                                                                             if(n.1t.ndiff) then                                                           goto 1000                                                                     endif                                                                         1250                                                                              continue                                                                  c                                                                             c                                                                                     jj=jj+1                                                                       write(*,130) (nbase(i),i=1,nsub),jj                                             do 1100 i=1,nsub                                                                mset (jj , i) =nbase (i)                                          1100          continue                                                        c                                                                             c                                                                             1000                                                                              continue                                                                  c                                                                             c                                                                             write (*,*)                                                                   130 format(10x,20(1x,i1),5x,i5)                                               write (*,*)                                                                   write(*,120) jj                                                               120 format(1x,'Number of words=',i5)                                          c                                                                             c                                                                             end                                                                           c                                                                             c           *********************************************                     c           *********************************************                     __________________________________________________________________________

    __________________________________________________________________________    APPENDIX Ic                                                                   Exemplary computer program for generating                                     minimally cross hybridizing sets                                              (double stranded tag/single stranded tag complement)                          __________________________________________________________________________    Program 3tagN                                                                 c     Program 3tagN generates minimally cross-hybridizing                     c       sets of duplex subunits given i) N--subunit length,                   c       and ii) an initial homopurine sequence.                               c   character*1 sub1 (20)                                                     integer*2 mset(10000,20), nbase(20)                                           c                                                                             write(*,*) 'ENTER SUBUNIT LENGTH'                                             read(*,100)nsub                                                               100 format(i2)                                                                c                                                                             write(*,*) 'ENTER SUBUNIT SEQUENCE a & g only'                                read(*,110) (sub1(k) ,k=1,nsub)                                               110 format(20a1)                                                              c                                                                             ndiff=10                                                                      c             Let a=1 and g=2                                                 c                                                                             do 800 kk=1,nsub                                                              if(sub1(kk) .eq. 'a') then                                                            mset (1, kk) =1                                                               endif                                                                               if(subl(kk) .eq. 'g') then                                                      mset (1,kk)=2                                                                 endif                                                         800 continue                                                                  c                                                                             jj=1                                                                          c                                                                             do 1000 k1=1,3                                                                        do 1000 k2=1,3                                                                  do 1000 k3=1,3                                                                  do 1000 k4=1,3                                                                  do 1000 k5=1,3                                                                  do 1000 k6=1,3                                                                  do 1000 k7=1,3                                                                  do 1000 k8=1,3                                                                  do 1000 k9=1,3                                                                  do 1000 k10=1,3                                       do 1000 k11=1,3                                                                       do 1000 k12=1,3                                                                 do 1000 k13=1,3                                                                 do 1000 k14=1,3                                                                 do 1000 k15=1,3                                                                 do 1000 k16=1,3                                                                 do 1000 k17=1,3                                                                 do 1000 k18=1,3                                                                 do 1000 k19=1,3                                                                 do 1000 k20=1,3                                       c                                                                                           nbase(1)=k1                                                                   nbase(2)=k2                                                                   nbase(3)=k3                                                                   nbase(4)=k4                                                                   nbase(5)=k5                                                                   nbase(6)=k6                                                                   nbase(7)=k7                                                                   nbase(8)=k8                                                                   nbase(9)=k9                                                                   nbase(10)=k10                                                                 nbase(11)=k11                                                                 nbase(12)=k12                                                                 nbase(13)=k13                                                                 nbase(14)=k14                                                                 nbase(15)=k15                                                                 nbase(16)=k16                                                                 nbase(17)=k17                                                                 nbase(18)=k18                                                                 nbase(19)=k19                                                                 nbase(20)=k20                                                   c                                                                                       do 1250 nn=1,jj                                                     c                                                                                       n=0                                                                           do 1200 j=1,nsub                                                                  if(mset(nn,j) .eq.1 .and. nbase(j) .ne.1 .or.                   1               mset(nn,j) .eg.2 .and. nbase(j) .ne.2 .or.                    2               mset(nn,j) .eq.3 .and. nbase(j) .ne.3 .or.                    3               mset(nn,j) .eq.4 .and. nbase(j) .ne.4) then                                   n=n+1                                                                         endif                                                         1200            continue                                                      c                                                                             if(n.1t.ndiff) then                                                                   goto 1000                                                                     endif                                                                 1250                                                                              continue                                                                  c                                                                                       jj=jj+1                                                                       write(*,130) (nbase(i) ,i=1,nsub) ,jj                                           do 1100 i=1,nsub                                                                mset(jj ,i)=nbase(i)                                            1100            continue                                                      c                                                                             1000                                                                              continue                                                                  c                                                                             write (*,*)                                                                   130   format(10x,20(1x,il) ,5x,i5)                                            write (*,*)                                                                   write(*,120) jj                                                               120   format(1x,'Number of words=',i5)                                        c                                                                             c                                                                             end                                                                           __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES:  27                                           - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 66 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              50NNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNTTT                      #    66            T                                                          - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 39 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #    39            NNNN NNNNNNNNNN NNNNNNTGG                                  - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #       11                                                                    - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 62 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              50NNNNNN NNNNNNNNNN NNNNNNNNNN NNNTTTTTTT                      #       62                                                                    - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 38 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #     38           ATCC ACTCGAGATC CCAATCCA                                   - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               NNNN                                                       - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24NNNN NNNN                                                  - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               GTNN                                                       - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 68 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              50NNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNGGT                      #  68              TT                                                         - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #       11                                                                    - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 43 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 43               GATT TGGTAAAGTA ATGTAAAGGA TTA                             - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 43 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 43               AGGA TTTGATAGTA TTTGTGATGA TTA                             - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 43 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 43               TTGA TTGTAAAAAG AAAGTTTGTT TGA                             - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 42 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #  42              NNNN NNNNNNNNNN NNNNNNNNNN NA                              - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 26 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              26  GCAT CGACCC                                                - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #        10                                                                   - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #        10                                                                   - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CGAT                                                       - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CGAT                                                       - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CGAT                                                       - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CGAT                                                       - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 44 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 44               NNNN NNNNNNNNNN NNNNNNNNNN CGGT                            - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               ACCG                                                       - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CGAT                                                       - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #25:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               ACCG                                                       - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               #26:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               CCCC                                                       - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 62 nucleoti - #des                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               #27:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              50TTGATA GAGAGGACCT GTTTAAACGG ATCCTCTTCC                      #       62                                                                    __________________________________________________________________________

I claim:
 1. A computer program product comprising:a computer usablestorage medium having computer readable code embodied therein forcausing a computer to generate a set of non-hybridizing oligonucleotidesof a given length, said computer readable code comprising: computerreadable program code configured to cause said computer to effect anassignment mechanism configured to assign a plurality of identifyingdata values representing different nucleotides; computer readableprogram code configured to cause said computer to effect a sequencegeneration mechanism configured to provide in a memory, from saidplurality of identifying data values, a data-value sequence in a firstset of possible sequences, where each possible sequence in said firstset of possible sequences represents an oligonucleotide having aspecified nucleotide at a specified residue position in saidoligonucleotide of said given length; computer readable program codeconfigured to cause said computer to effect a sequence comparisonmechanism configured to compare in said memory said data-value sequencefrom said first set of possible sequences with a data-value sequence ina second set of possible sequences, on the basis of number of positionsat which said data-value sequence from said first set of possiblesequences and said data-value sequence in said second set of possiblesequences have different data values; computer readable program codeconfigured to cause said computer to effect a sequence inclusionmechanism configured to add said data-value sequence from said first setof possible sequences to said second set of possible sequences if thesequence comparison mechanism determined that said data-value sequencefrom said first set of possible sequences and said data-value sequencein said second set of possible sequences differ by at least N positions,where N is a specified value greater than one; computer readable programcode configured to cause said computer to effect a sequence paircomparison mechanism configured to compare each pair of data-valuesequences in said second set of possible sequences on the basis ofnumber of positions at which each data-value sequence in said pair ofdata-value sequences have different data values; computer readableprogram code configured to cause said computer to effect a sequenceretention mechanism configured to retain in said second set of possiblesequences, data-value sequences that differ by said at least N positionsfrom all other data-value sequences in said second set of possiblesequences; and computer readable program code configured to cause saidcomputer to effect a first iterative mechanism configured torepetitively invoke the sequence generation mechanism, the sequencecomparison mechanism, the sequence inclusion mechanism, the sequencepair comparison mechanism and the sequence retention mechanism until adesired number of data-value sequences have been retained within saidsecond set of possible sequences.
 2. The computer program product ofclaim 1 wherein said first set of possible sequences includespermutations of data-value sequences having said given length.
 3. Thecomputer program product of claim 1 wherein the sequence generationmechanism generated a list of possible data-value sequences of saidgiven length and the sequence generation mechanism furthercomprises:computer readable program code configured to cause saidcomputer to effect a selection mechanism configured to select saiddata-value sequence from said list of possible data-value sequences. 4.The computer program product of claim 1 wherein:the sequence generationmechanism includes computer readable program code configured to causesaid computer to effect a selection mechanism configured to arbitrarilyselect said data-value sequence from said first set of possiblesequences; if said data-value sequence has been placed in said secondset of possible sequences by the subsequent sequence generationmechanism, the sequence inclusion mechanism includes computer readableprogram code configured to cause said computer to effect a setcomparison mechanism configured to compare said data-value sequence withall other data-values sequences within said second set of possiblesequences; and the sequence retention mechanism is configured to retainsaid data-value sequence within said second set of possible sequencesonly if the set comparison mechanism determines that said data-valuesequence differs in data values from all other sequences in said secondset of possible sequences by said at least N positions.
 5. The computerprogram product of claim 3 that further includes computer readableprogram code configured to cause said computer to effect a seconditerative mechanism configured to repetitively invoke the sequencegeneration mechanism, the sequence comparison mechanism, the sequenceinclusion mechanism, the sequence pair comparison mechanism and thesequence retention mechanism until each data-value sequence in saidfirst set of possible sequences has been compared with at least onedata-value sequence in said second set of possible sequences.